A bacteriophage lambda vector for cloning with BamHI and Sau3A

Mizusawa, Saeko; Ward, D.

doi:10.1016/0378-1119(82)90200-1

Cited by 75 publications

(40 citation statements)

References 12 publications

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“…In addition to the able within 30 NfrC protein, at least three other gene products are required ractionation of for N4 adsorption. nfrA encodes an outer membrane protein )f the overexwhich is presumably the receptor, nfrB encodes an inner ise, particulate membrane protein (20), while the gene product of nfrD has not shown).…”

Section: Resultsmentioning

confidence: 99%

A cytoplasmic protein, NfrC, is required for bacteriophage N4 adsorption

et al. 1993

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At least four genes are required for irreversible adsorption of bacteriophage N4. nfrA and nfrB have been characterized previously and encode an outer membrane protein and inner membrane protein, respectively. The nfrC gene product is characterized in detail in this study. We have mapped the nfrD locus to min 52 on the Escherichia coli linkage map. Maxicell analysis of nfrC and a null allele (nfrC2) cloned into a high-copy-number plasmid shows its gene product to be 42 kDa in size. We determined the nfrC nucleotide sequence which predicts a gene product of 42 kDa. Western blots (immunoblots) of Escherichia coli proteins after cellular fractionation show NfrC to be a cytoplasmic protein which is required for irreversible bacteriophage N4 adsorption, an event occurring at the cell surface.Bacteriophage N4 is a lytic phage specific for Escherichia coli K-12 (19). It is unique among the DNA-containing bacteriophages because transcription of the early region of the genome is independent of the activity of the host RNA polymerase. Instead, transcription of the early phage genes is carried out by a virion-encapsulated DNA-dependent RNA polymerase (12). Therefore, the first event in N4 infection requires the injection of both its 72-kbp double-stranded DNA genome and the virion RNA polymerase, a large single-subunit enzyme (M,, 320,000). We have been conducting a genetic analysis of N4 adsorption to learn more about E. coli membrane structure and the penetration of large macromolecular species such as the phage genome and RNA polymerase through the outer and inner membranes.Selection for spontaneously occurring mutations which confer N4 resistance has consistently resulted in mutations mapping to four different loci (20), suggesting the involvement of several E. coli gene products and a relatively complex process. Approximately 60% of the mutants map to nfrA and nfrB, two tightly linked loci at 12 min on the E. coli linkage map (2) whose coding regions overlap (21). These genes have been previously characterized as an outer membrane protein (NfrA) which presumably is the structural receptor for N4 and an inner membrane protein (NfrB). The remaining mutations map to two unlinked loci, nfrC at 85 min and nfrD, which, until this study, had not been precisely mapped.In this article, we characterize the nfrC gene product in detail and present its cloning and the determination of its nucleotide sequence. MATERIALS AND METHODSBacterial strains. (29,38).Cloning nfrC. We screened a XD69 (30) library of E. coli DNA (generously provided by S. Gottesman) for potential clones of nfrC by using the approach described previously (20). We plated the library for single plaques on a lawn of strain KW8801 (carrying the nfrC2 mutation) which had been seeded with N4. Clear plaques representing phage potentially complementing the adsorption defect were picked and purified. Thosc that were turbid in the absence of N4 after replating were lysogenized into KW8801 and tested for their ability to complement the nfrC2 mutation and to integrate at ...

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Section: Resultsmentioning

confidence: 99%

A cytoplasmic protein, NfrC, is required for bacteriophage N4 adsorption

et al. 1993

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“…Standard cloning methods were as described previously (33). XPR6 was isolated from an E. coli library, constructed by introducing fragments from a partial Sau3A digest of the E. coli genome into the BamHI site of the XD69 vector (37). pPR6L contains the 3.5-kb BglII-HindIII fragment of the XPR6 insert cloned in HindIII-BamHI-digested pGEM-3 (Promega Biotec); pPR6M contains the 1.5-kb AccI-HindIII fragment (AccI end filled in with Klenow DNA polymerase) of the XPR6 insert cloned in pGEM-3 digested with HindIII and SmaI; pPR6S contains the 1-kb HindIII-EcoRI fragment of the XPR6 insert cloned in pGEM-3 digested with the same enzymes.…”

Section: Methodsmentioning

confidence: 99%

Isolation, characterization, and sequence of an Escherichia coli heat shock gene, htpX

et al. 1991

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We isolated and characterized a new Escherichia coli gene, htpX. The htpX gene has been localized at min 40.3 on the chromosome. We determined its transcription and translation start site. htpX expresses a 32-kDa protein from a monocistronic transcript; expression of this protein is induced by temperature upshift. htpX is expressed from a or32-dependent promoter and is thus part of the heat shock regulon. Cells carrying a htpX gene disruption grow well at all temperatures and under all conditions tested and have no apparent phenotype. However, cells which overexpress a truncated form of the protein display a higher rate of degradation of puromycyl peptides.The heat shock response consists in a transient increase, after temperature upshift, in the synthesis of a small subset of proteins, the heat shock proteins (hsps). This response has been observed in all cells so far examined (reviewed in reference 31). In Escherichia coli, temperature upshift, as well as other treatments such as UV irradiation, bacteriophage infection, and overproduction of abnormal proteins, leads to the preferential synthesis of at least 17 hsps (reviewed in reference 38). The central regulator of the heat shock response in E. coli is the heat shock gene-specific RNA polymerase subunit, cr32 (22, 23).Some hsps have been highly conserved throughout evolution. Three E. coli hsps, GroEL, DnaK, and C62.5, are homologous to the mitochondrial HSP58 protein and to the eukaryotic HSP70 and HSP90 protein families, respectively (5, 6, 35). In contrast, the small hsps, although often conserved within the same organism, display little similarity between different organisms (32).GroE is involved in the assembly of multiprotein complexes (15,20), and DnaK, DnaJ, and GrpE have been shown to participate in the refolding of denatured proteins (14). In addition, strains carrying mutations in these genes are defective in the proteolysis of abnormal proteins (48). The Lon protease, which degrades abnormal proteins in the cell (21, 34), is a heat shock protein as well (16). A common denominator of most conditions that induce the synthesis of the hsps is thought to be the appearance of unfolded proteins in the cell (4,17,41). It is possible that the primary role of the heat shock response is to remove unfolded proteins from the cell, either by refolding or by degrading them.The lysogenization process of bacteriophage A involves several phage proteins, such as the CII transcriptional activator, which is required for the initiation of transcription of the repressor and integrase genes (45), and the CIII protein, whose role is to stabilize CII (3, 24). Several host genes, e.g., rnc (2) and hfl (7), are involved in this process as well. We were interested in isolating additional host genes participating in the regulation of phage lysogenization.A screening procedure aimed at isolating such genes from

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“…XD69 is a X imm21 cloning vector (10 (4). Gal' revertants of N6345 appeared at low frequency, presumably arising as the result of recombination between the duplicated sequences.…”

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Stimulation of IS1 excision by bacteriophage P1 ref function

Gottesman

1989

J Bacteriol

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Lysogenization by a clts variant of coliphage P1, Plcl.100, markedly increased the frequency of reversion of a gaiT::ISI mutation. The formation of Gal' colonies presumably occurs by microhomologous recombination between the 9-base-pair repeats in gafT (CGCCGCTAC) Several temperate coliphages are known to express recombination functions. The int and xis genes of lambda, for example, specify site-specific recombination enzymes, while the reda and redb gene products, X exonuclease and beta protein, respectively, promote homologous recombination. The four genes are clustered in the lambda genome, and their roles in lambda physiology are known; the first two are required for prophage integration and excision, whereas the products of the red genes convert lambda monomers into packageable multimers.Prophage P1 expresses a site-specific recombination function, Cre, which improves P1 plasmid maintenance by cutting and rejoining P1 chromosomes at the P1 lox sites (5,16,18). RecA is responsible for most P1 circularization, since Cre can circularize only those P1 molecules, about 20% of the total, which bear redundant lox sites. In recA mutants, however, Cre is essential for P1 lysogenization and lytic growth.The lytic development of P1 prophage is blocked by the bacteriophage-encoded cI gene product. Lysogens carrying Plcl. 100, a clts mutant, are stable at 32°C but are induced at temperatures above 39°C (13). Several years ago, we observed that Plcl.100 lysogens displayed a phenotype related to recombination. Escherichia coli bearing the IS] gal operon insertion, galTN102 (6), revert to Gal' at elevated frequencies when lysogenic for Plclts. We assume that this reversion results from recombination between the repeated 9-base-pair (bp) galT target sequences which flank the IS] transposon, a process referred to as precise excision. Independently, Windle and Hays (19) noted that recombination between two close but nonoverlapping deletions in lacZ was likewise increased by Plcl.100 prophage. Plcl.100 did not appear to stimulate recombination within large regions of DNA homology (19). These observations suggested that P1 carries a recombination enhancement function (re]) which

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A bacteriophage lambda vector for cloning with BamHI and Sau3A

Cited by 75 publications

References 12 publications

A cytoplasmic protein, NfrC, is required for bacteriophage N4 adsorption

A cytoplasmic protein, NfrC, is required for bacteriophage N4 adsorption

Isolation, characterization, and sequence of an Escherichia coli heat shock gene, htpX

Stimulation of IS1 excision by bacteriophage P1 ref function

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