Showing papers on "Lambda phage published in 1989"

New recA mutations that dissociate the various RecA protein activities in Escherichia coli provide evidence for an additional role for RecA protein in UV mutagenesis.

Abstract: To isolate strains with new recA mutations that differentially affect RecA protein functions, we mutagenized in vitro the recA gene carried by plasmid mini-F and then introduced the mini-F-recA plasmid into a delta recA host that was lysogenic for prophage phi 80 and carried a lac duplication. By scoring prophage induction and recombination of the lac duplication, we isolated new recA mutations. A strain carrying mutation recA1734 (Arg-243 changed to Leu) was found to be deficient in phi 80 induction but proficient in recombination. The mutation rendered the host not mutable by UV, even in a lexA(Def) background. Yet, the recA1734 host became mutable upon introduction of a plasmid encoding UmuD*, the active carboxyl-terminal fragment of UmuD. Although the recA1734 mutation permits cleavage of lambda and LexA repressors, it renders the host deficient in the cleavage of phi 80 repressor and UmuD protein. Another strain carrying mutation recA1730 (Ser-117 changed to Phe) was found to be proficient in phi 80 induction but deficient in recombination. The recombination defect conferred by the mutation was partly alleviated in a cell devoid of LexA repressor, suggesting that, when amplified, RecA1730 protein is active in recombination. Since LexA protein was poorly cleaved in the recA1730 strain while phage lambda was induced, we conclude that RecA1730 protein cannot specifically mediate LexA protein cleavage. Our results show that the recA1734 and recA1730 mutations differentially affect cleavage of various substrates. The recA1730 mutation prevented UV mutagenesis, even upon introduction into the host of a plasmid encoding UmuD* and was dominant over recA+. With respect to other RecA functions, recA1730 was recessive to recA+. This demonstrates that RecA protein has an additional role in mutagenesis beside mediating the cleavage of LexA and UmuD proteins.

Discovery of a protein phosphatase activity encoded in the genome of bacteriophage lambda. Probable identity with open reading frame 221.

Abstract: Infection of Escherichia coli with phage lambda gt10 resulted in the appearance of a protein phosphatase with activity towards 32P-labelled casein. Activity reached a maximum near the point of cell lysis and declined thereafter. The phosphatase was stimulated 30-fold by Mn2+, while Mg2+ and Ca2+ were much less effective. Activity was unaffected by inhibitors 1 and 2, okadaic acid, calmodulin and trifluoperazine, distinguishing it from the major serine/threonine-specific protein phosphatases of eukaryotic cells. The lambda phosphatase was also capable of dephosphorylating other substrates in the presence of Mn2+, although activity towards 32P-labelled phosphorylase was 10-fold lower, and activity towards phosphorylase kinase and glycogen synthase 25 50-fold lower than with casein. No casein phosphatase activity was present in either uninfected cells, or in E. coli infected with phage lambda gt11. Since lambda gt11 lacks part of the open reading frame (orf) 221, previously shown to encode a protein with sequence similarity to protein phosphatase-1 and protein phosphatase-2A of mammalian cells [Cohen, Collins, Coulson, Berndt & da Cruz e Silva (1988) Gene 69, 131-134], the results indicate that ORF221 is the protein phosphatase detected in cells infected with lambda gt10. Comparison of the sequence of ORF221 with other mammalian protein phosphatases defines three highly conserved regions which are likely to be essential for function. The first of these is deleted in lambda gt11.

Cloning, sequencing, and mapping of the bacterioferritin gene (bfr) of Escherichia coli K-12.

Abstract: The bacterioferritin (BFR) of Escherichia coli K-12 is an iron-storage hemoprotein, previously identified as cytochrome b1. The bacterioferritin gene (bfr) has been cloned, sequenced, and located in the E. coli linkage map. Initially a gene fusion encoding a BFR-lambda hybrid protein (Mr 21,000) was detected by immunoscreening a lambda gene bank containing Sau3A restriction fragments of E. coli DNA. The bfr gene was mapped to 73 min (the str-spc region) in the physical map of the E. coli chromosome by probing Southern blots of restriction digests of E. coli DNA with a fragment of the bfr gene. The intact bfr gene was then subcloned from the corresponding lambda phage from the gene library of Kohara et al. (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987). The bfr gene comprises 474 base pairs and 158 amino acid codons (including the start codon), and it encodes a polypeptide having essentially the same size (Mr 18,495) and N-terminal sequence as the purified protein. A potential promoter sequence was detected in the 5' noncoding region, but it was not associated with an "iron box" sequence (i.e., a binding site for the iron-dependent Fur repressor protein). BFR was amplified to 14% of the total protein in a bfr plasmid-containing strain. An additional unidentified gene (gen-64), encoding a relatively basic 64-residue polypeptide and having the same polarity as bfr, was detected upstream of the bfr gene.

Differential regulation of lambda pL and pR promoters by a cI repressor in a broad-host-range thermoregulated plasmid marker system.

Abstract: Plasmid systems with unique markers were constructed to assess the fate of recombinant DNA and genetically manipulated bacteria in soil and freshwater model environments. On such constructs the marker gene, xylE (for catechol 2,3-dioxygenase), is expressed from the lambda promoter pL or pR, each of which is controlled by the temperature-sensitive lambda repressor c1857. Combinations of these elements were cloned into the broad-host-range plasmid pKT230 to form pLV1010 (pL-xylE), pLV1011 (pL-xylE-c1857), and pLV1013 (pR-xylE-c1857). The recombinant plasmids were introduced into different gram-negative bacteria. The thermoregulated system of pLV1013 functioned well in a range of species, with xylE induction being readily achieved by elevation of the temperature from 28 to 37 degrees C. There was a difference in the induction of catechol 2,3-dioxygenase activity, depending on whether xylE was expressed from pL (pLV1011) or pR (pLV1013). Our observations on testing the different systems in a number of hosts suggest that genes carried by the DNA of genetically engineered microorganisms may not be expressed in a predictable manner following transfer from the release host to other species.

Phasing of protein-induced DNA bends in a recombination complex

Abstract: Many of the structures responsible for replication, transcription initiation and recombination arise from complex sets of protein-protein interactions and the folding of DNA in three dimensions, with protein-induced bending of DNA often playing an integral role. The magnitude and orientation of DNA bending induced by various single proteins has been estimated by gel mobility shift methods and by modelling of crystallographic data. The site-specific recombination by which bacteriophage lambda (phage lambda) integrates into the chromosome of its host Escherichia coli requires a host protein, 'integration host factor' (IHF), which is known to be able to bend the DNA to which it binds. To determine the three-dimensional path of DNA within the higher order structure responsible for phage lambda site-specific recombination, we have determined the relative direction of IHF-induced bending at each of the three binding sites within the complex. IHF, which appears to bend DNA by more than 140 degrees, is a major determinant of the DNA path in the recombination complex and is also involved in a wide range of other cellular events.

Vaccinia DNA topoisomerase I promotes illegitimate recombination in Escherichia coli.

Abstract: Vaccinia virus encapsidates a Mr 32,000 type IDNA topoisomerase. Although the vaccinia gene encoding the topoisomerase is essential for virus growth, the role of the enzyme in vivo remains unclear. In the present study, the physiologic consequences of vaccinia topoisomerase action have been examined in a heterologous system, Escherichia coli. The vaccinia topoisomerase gene was inducibly expressed in an int-lambda lysogen BL21(DE3) using a T7 RNA polymerase-based transcription system. Expression of active topoisomerase in this context resulted in recA-dependent lysogenic induction as well as cell lysis. Surprisingly, topoisomerase expression also effected a 200-fold increase in the titer of infectious lambda phage, apparently by promoting int-independent prophage excision. This effect was not observed during lysogenic induction with nalidixic acid. Restriction analysis of genomic DNA from plaque-purified excisants revealed (in 10 of 10 cases) gross alterations of the DNA structure around the att site relative to the structure of the parental phage DE3. It is construed therefore that vaccinia DNA topoisomerase I acts to promote illegitimate recombination in E. coli.

Interaction between the sbcC gene of Escherichia coli and the gam gene of phage lambda.

Abstract: gam mutants of phage lambda carrying long palindromes fail to form plaques on wild-type Escherichia coli but do grow on strains that are mutant in the sbcC gene. gam + lambda carrying the same palindrome grow on both hosts and on a host deleted for the recB, C and D genes. These results suggest that the Gam protein of lambda, known to interact also with E. coli's recBCD protein, can interact with the product of the sbcC gene.

A component of the side tail fiber of Escherichia coli bacteriophage lambda can functionally replace the receptor-recognizing part of a long tail fiber protein of the unrelated bacteriophage T4.

Abstract: The distal part of the long tail fiber of Escherichia coli bacteriophage T4 consists of a dimer of protein 37. Dimerization requires the catalytic action of protein 38, which is encoded by T4 and is not present in the virion. It had previously been shown that gene tfa of the otherwise entirely unrelated phage lambda can functionally replace gene 38. Open reading frame (ORF) 314, which encodes a protein that exhibits homology to a COOH-terminal area of protein 37, is located immediately upstream of tfa. The gene was cloned and expressed in E. coli. An antiserum against the corresponding polypeptide showed that it was present in phage lambda. The serum also reacted with the long tail fibers of phage T4 near their free ends. An area of the gene encoding a COOH-terminal region of ORF 314 was recombined, together with tfa, into the genome of T4, thus replacing gene 38 and a part of gene 37 that codes for a COOH-terminal part of protein 37. Such T4-lambda hybrids, unlike T4, required the presence of outer membrane protein OmpC for infection of E. coli B. An ompC missense mutant of E. coli K-12, which was still sensitive to T4, was resistant to these hybrids. We conclude that the ORF 314 protein represents a subunit of the side tail fibers of phage lambda which probably recognize the OmpC protein. ORF 314 was designated stf (side tail fiber). The results also offer an explanation for the very unusual fact that, despite identical genomic organizations, T4 and T2 produce totally different proteins 38. An ancestor of T4 from the T2 lineage may have picked up tfa and stf from a lambdoid phase, thus possibly demonstrating horizontal gene transfer between unrelated phage species.

Cloning and DNA sequence of plasmid determinant iss , coding for increased serum survival and surface exclusion, which has homology with lambda DNA

Abstract: Escherichia coli K12 cells carrying a cloned 1.4 kb HindIII fragment from plasmid ColV2-K94, showed increased survival in guinea pig serum. The recombinant plasmid also conferred group II surface exclusion, i.e. the cells were reduced in recipient ability towards the incoming plasmid R538drd in conjugation experiments. Southern blotting suggested homology with bacteriophage lambda DNA and to the insertion element IS2. Determination of the DNA sequence of the fragment demonstrated the presence of a truncated IS2 (165 bp), separated by 250 bp from a 900 bp stretch of homology with lambda DNA, beginning within the Rz gene and continuing in the rightward direction on the lambda map. A 97 amino acid open reading frame (ORF) adjacent to Rz and on the opposite strand, remained intact in iss, with several amino acid changes. The ORF in iss is preceded by sequences resembling prokaryotic ribosome binding sites and promoters.

Tn5supF, a 264-base-pair transposon derived from Tn5 for insertion mutagenesis and sequencing DNAs cloned in phage lambda.

Abstract: We constructed a derivative of transposon Tn5 called Tn5supF for insertion mutagenesis and sequencing DNAs cloned in phage lambda. This element carries a supF amber-suppressor tRNA gene. Its insertion into lambda can be selected by plaque formation by using nonsuppressing (sup0) Escherichia coli for amber mutant lambda phage and sup0 dnaB-amber E. coli for nonamber lambda phage. Tn5supF is just 264 base pairs long. It transposes efficiently and inserts quasi-randomly into DNA targets. The unique sequences near its termini can be used as primer binding sites for dideoxynucleotide DNA sequencing, thus permitting the direct sequencing of DNAs cloned in phage lambda without subcloning.

Cloning and expression of the streptococcal C5a peptidase gene in Escherichia coli: linkage to the type 12 M protein gene.

Abstract: A genomic library of Streptococcus pyogenes CS24 DNA was constructed by cloning streptococcal DNA partially digested with Sau3A into the lambda replacement vector EMBL3. The expression of streptococcal C5a peptidase (SCP) was analyzed by radioimmunoassay with hyperimmune rabbit serum. Two clones, lambda 4.1 and lambda 4.2, were found to express the desired antigen, and various DNA fragments from the hybrid bacteriophage lambda 4.1 were subcloned into the plasmid vector pUC9 in Escherichia coli. One of the recombinant plasmids, designated pTT1, contained a 5.8-kilobase (kb) streptococcal DNA insert. Analysis of total cellular protein from this E. coli clone by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western (immuno-) blotting identified a 140,000-Mr protein, similar in size to the native protein purified from S. pyogenes. Cloned SCP was functionally active, as shown by its ability to inhibit C5a-mediated chemotaxis. By deletion analysis with both restriction endonucleases and BAL 31 nuclease, the SCP gene was localized to a 4.3-kb segment of DNA. Southern hybridization experiments showed that the type 12 M protein-coding sequence is also present in the hybrid phage lambda 4.1, at approximately 2 kb upstream of the SCP structural gene. Western blot analysis indicated that the cloned streptococcal DNA in lambda 4.1 directed the expression of both SCP and M12 protein. Images

Modulation of the SOS response by truncated RecA proteins.

Abstract: RecA protein plays several key roles in the SOS response. We have constructed truncated proteins and examined their capacity to accomplish Weigle reactivation and mutagenesis of bacteriophage lambda and recombination in Escherichia coli. Our data indicate that the 17 carboxyl terminal amino acids are not essential to RecA function. However in the presence of wild-type RecA protein, the truncated protein reduces the efficiency of recombination without affecting either mutagenesis or induction of an SOS gene or Weigle reactivation. The data presented here suggest that activation of RecA protein does not involve mixed multimers or is not affected by their presence.

The Escherichia coli protein, Fis: specific binding to the ends of phage Mu DNA and modulation of phage growth.

Abstract: We show, using gel retardation, that crude Escherichia coli cell extracts contain a protein which binds specifically to DNA fragments carrying either end of the phage Mu genome. We have identified this protein as Fis, a factor involved in several site-specific recombinational switches. Furthermore, we show that induction of a Mucts62 prophage in a fis lysogen occurs at a lower temperature than that of a wild-type strain, and that spontaneous induction of Mucts62 is increased in the fis mutant. DNasel footprinting using either crude extracts or purified Fis indicate that binding on the left end of Mu occurs at a site which overlaps a weak transposase binding site. Thus, Fis may modulate Mu growth by influencing the binding of transposase, or other proteins, to the transposase binding site(s), in a way similar to its influence on Xis binding in phage lambda.

Overexpression of N antitermination proteins of bacteriophages lambda, 21, and P22: loss of N protein specificity.

Abstract: The N protein of bacteriophage lambda (N lambda) modifies Escherichia coli RNA polymerase in such a way that it transcribes through termination signals, a process called antitermination. N antitermination normally occurs only if the template contains a specific utilization or nut site upstream of the terminators and only in the presence of host-encoded Nus proteins. The lambda-related phages 21 and P22 produce N analogs, N21 and N22, but these require different nut sites and show a different pattern of functional interaction with one of the Nus factors, NusA, according to whether this protein is of E. coli or Salmonella origin (NusAEc or NusASal). We report the overproduction of N lambda, N21, or N22, each of which was induced by isopropyl-beta-D-thiogalactopyranoside at 37 degrees C from its cloned position downstream from ptac on a high-expression plasmid, each in a host that provided NusAEc or NusASal. Overproduction of each of these N proteins resulted in relaxed specificity for nut, which was shown by the ability to complement N mutants of heterologous phages; NusA specificity was determined by the N type that was present in these complementation tests. We also observed that excess N was able to suppress transcriptional polarity in the particular case of cloned 'trpA, the last gene of the tryptophan operon, although there was no effect on polarity within chromosomal trpE. Such polarity is attributed to the presence of cryptic intragenic terminators that become exposed in the absence of translation. Because there is no known nut site cis to 'trpA, we suggest that the 'trpA segment itself fortuitously contains a nut sequence that is able to function with excess N of any of the types tested and with either NusAEc or NusASal. We also found that excess N of any specificity, or even inactive N with missense mutation, could cause an increase in the level of NusAEc or NusASal, possibly because interaction between N and NusA, but independent of nut, whether functional or not, interferes with the autoregulation of NusA synthesis. These observations highlight the importance of protein concentration for the specificity of interactions both with other proteins and with nucleic acids. They also indicate that the interaction between N and NusA requires nut participation both for specificity and functionality.

The use of λplac-Mu hybrid phages in Klebsiella pneumoniae and the isolation of stable Hfr strains

Abstract: Klebsiella pneumoniae 1033-5P14 and its P1-sensitive derivative KAY2026 were found to be resistant to λ although they contained a LamB protein, active as a maltoporin. Sensitive derivatives could only be obtained after introduction of the pTROY9 plasmid which expresses lamB and the corresponding λ receptor from Escherichia coli K12 at high levels. Lysogenic derivatives from such strains were shown to carry the phage at secondary att sites and to give high titer lysates when induced. The use of λplac-Mu hybrid phages allowed the isolation from several operons of lacZ fusions orientated in, or against, the direction of transcription. Such insertions could subsequently be used to isolate stable Hfr strains by allowing homologous recombination to take place between the lac genes in the inserted hybrid phages and those of plasmid F′ts114 lac+ zzf20::Tn10. The Hfr strains were able to transfer K. pneumoniae chromosomal genes and allowed the mapping of such genes. Characteristic differences between this conjugation system and that of Escherichia coli K12 are discussed. The insertions also allowed determination of the direction of transcription of the gut gene, the newly mapped scr gene and of the sor gene cluster encoding enzymes for the metabolism of d-glucitol, sucrose and l-sorbose.

Lambda phage shuttle vectors for analysis of mutations in mammalian cells in culture and in transgenic mice.

Abstract: Foreign DNA sequences contained in lambda bacteriophage genomes integrated in mammalian DNA can be efficiently rescued into infectious phage particles by treatment of the mammalian DNA with lambda-packaging extracts prepared in E. coli. This system provides for rapid, non-selective recovery of stably integrated, chromosomal sequences into lambda phage for subsequent analysis in bacterial systems. Since rescue is prior to selection, mutations can be recovered from intact animals made transgenic for the phage-target gene sequences. Such approaches allow study of physiologically relevant aspects of mammalian mutagenesis at the molecular level.

Retroregulation of the bacteriophage lambda int gene: limited secondary degradation of the RNase III-processed transcript.

Abstract: Expression of the int gene of bacteriophage lambda from two promoters, pI and pL, is differentially regulated through RNA processing. Efficient Int protein synthesis from the pL RNA is inhibited by the action of sib, a cis-acting retroregulator downstream from the int gene. We have used mapping procedures with nuclease S1 to study the pL transcripts produced in vivo after phage lambda infection. We have found an RNase III-dependent processing site within the Int coding sequence, 387 nucleotides upstream from the site of the primary cleavage by RNase III at Sib. This secondary processing site is located at the most stable region of secondary structure in the sib int region, as predicted by computer analysis. We suggest that RNase III cleavage at the Sib site allows processive exonucleolytic degradation of the RNA to proceed to a region of secondary structure within the Int coding sequence, which protects the upstream region of the transcript from further degradation.

Translesion synthesis is the main component of SOS repair in bacteriophage lambda DNA.

Abstract: Agents that interfere with DNA replication in Escherichia coli induce physiological adaptations that increase the probability of survival after DNA damage and the frequency of mutants among the survivors (the SOS response). Such agents also increase the survival rate and mutation frequency of irradiated bacteriophage after infection of treated bacteria, a phenomenon known as Weigle reactivation. In UV-irradiated single-stranded DNA phage, Weigle reactivation is thought to occur via induced, error-prone replication through template lesions (translesion synthesis [P. Caillet-Fauquet, M: Defais, and M. Radman, J. Mol. Biol. 117:95-112, 1977]). Weigle reactivation occurs with higher efficiency in double-stranded DNA phages such as lambda, and we therefore asked if another process, recombination between partially replicated daughter molecules, plays a major role in this case. To distinguish between translesion synthesis and recombinational repair, we studied the early replication of UV-irradiated bacteriophage lambda in SOS-induced and uninduced bacteria. To avoid complications arising from excision of UV lesions, we used bacterial uvrA mutants, in which such excision does not occur. Our evidence suggests that translesion synthesis is the primary component of Weigle reactivation of lambda phage in the absence of excision repair. The greater efficiency in Weigle reactivation of double-stranded DNA phage could thus be attributed to some inducible excision repair unable to occur on single-stranded DNA. In addition, after irradiation, lambda phage replication seems to switch prematurely from the theta mode to the rolling circle mode.

Phage genetic sites involved in lambda growth inhibition by the Escherichia coli rap mutant.

Abstract: The rap mutation of Escherichia coli prevents the growth of bacteriophage lambda. We have isolated phage mutants that compensate for the host deficiency. The mutations, named bar, were genetically located to three different loci of the lambda genome: barI in the attP site, barII in the cIII ea10 region, and barIII within or very near the imm434 region. The level of lambda leftward transcription correlates with rap exclusion. Phage lambda mutants partially defective in the pL promoter or in pL-transcript antitermination showed a Bar- phenotype. Conversely, mutants constitutive for transcription from the pI or pL promoters were excluded more stringently by rap bacteria. We conclude that rap exclusion depends on the magnitude of transcription through the wild type bar loci in the phage genome.

Suppression of recA deficiency in plasmid recombination by bacteriophage lambda beta protein in RecBCD- ExoI- Escherichia coli cells.

Abstract: Plasmid recombination, like other homologous recombination in Escherichia coli, requires RecA protein in most conditions. We have found that the plasmid recombination defect in a recA mutant can be efficiently suppressed by the beta protein of bacteriophage lambda. beta protein is required for homologous recombination of lambda chromosomes during lytic phage growth in a recA host and is known to have a strand-annealing activity resembling that of RecA protein. The bioluminescence recombination assay was used for genetic analysis of beta-protein-mediated plasmid recombination. Efficient suppression of the recA mutation by beta protein required the absence of the E. coli nucleases exonuclease I and RecBCD nuclease. These nucleases inhibit a RecA-mediated plasmid recombination pathway that is more efficient than the pathway functioning in wild-type cells. Like RecA-mediated plasmid recombination in RecBCD- ExoI- cells, beta-protein-mediated plasmid recombination depended on concurrent DNA replication and on the activity of the recQ gene. However, unlike RecA-mediated plasmid recombination, beta-protein-mediated recombination in RecBCD- ExoI- cells was independent of recF and recJ activities. We propose that inactivation of exonuclease I and RecBCD nuclease stabilizes a recombination intermediate that is involved in RecA- and beta-protein-catalyzed homologous pairing reactions. We suggest that the intermediate may be linear plasmid DNA with a protruding 3' end, since these nucleases are known to interfere with the synthesis of such linear forms. The different recF and recJ requirements for beta-protein-dependent and RecA-dependent recombinations imply that the mechanisms of formation or processing of the putative intermediate differ in the two cases.

Transcription of a region downstream from λ ori is required for replication of plasmids derived from coliphage lambda

Abstract: DNA replication of lambda phage depends on transcriptional activation at or around the λori region by RNA polymerase. To elucidate the function of the transcriptional activation, we constructed several plasmids carrying λori and lacP, whose relative locations and directions were different from each other, and studied replication activity of these recombinant plasmids. Transcription in a region immediately downstream from λori, but not in the λori region, was found to be essential for plasmid replication. Transcription proceeding over a certain minimal length was required and only rightward-directed transcription was effective for the activation