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PubMed1997

The hydrophilic C-terminal part of the lambda S holin is non-essential for intermolecular interactions.

Available evidence indicates that oligomerization of the bacteriophage lambda S holin leads to a non-specific lesion in the cytoplasmic membrane which permits transit of the phage encoded transglycosylase to the periplasm. In an attempt to locate an intermolecular interaction domain in S a chimeric protein comprising the N-terminal 32 aa of phage PhiX174 lysis protein E and the last 75 aa of lambda S has been constructed. We report that the E phi S fusion protein is stable, membrane bound, and inhibits S-mediated lysis in trans. C-terminal truncations of the E phi S fusion protein indicated that the hydrophilic C-terminal end of S (i.e. the last 15 aa) is non-essential for oligomerization.

Amino Acid Sequence↗

PubMed2006

Use of the lambda Red recombinase system to produce recombinant prophages carrying antibiotic resistance genes.

BACKGROUND: The Red recombinase system of bacteriophage lambda has been used to inactivate chromosomal genes in E. coli K-12 through homologous recombination using linear PCR products. The aim of this study was to induce mutations in the genome of some temperate Shiga toxin encoding bacteriophages. When phage genes are in the prophage state, they behave like chromosomal genes. This enables marker genes, such as antibiotic resistance genes, to be incorporated into the stx gene. Once the phages' lytic cycle is activated, recombinant Shiga toxin converting phages are produced. These phages can transfer the marker genes to the bacteria that they infect and convert. As the Red system's effectiveness decreased when used for our purposes, we had to introduce significant variations to the original method. These modifications included: confirming the stability of the target stx gene increasing the number of cells to be transformed and using a three-step PCR method to produce the amplimer containing the antibiotic resistance gene. RESULTS: Seven phages carrying two different antibiotic resistance genes were derived from phages that are directly involved in the pathogenesis of Shiga toxin-producing strains, using this modified protocol. CONCLUSION: This approach facilitates exploration of the transduction processes and is a valuable tool for studying phage-mediated horizontal gene transfer.

Algorithms↗

PubMed1988

Identification of the DNA binding domain of the phage lambda cII transcriptional activator and the direct correlation of cII protein stability with its oligomeric forms.

The bacteriophage lambda transcriptional activator protein cII is a DNA-binding protein that coordinately regulates transcription from phage promoters important for lysogenic growth. We have genetically and structurally characterized more than 80 different single amino acid substitutions in this 97-amino-acid protein. A subset of 25 of these variant proteins was utilized for detailed biochemical analysis, which allows us to define specific domains critical for sequence-selective DNA recognition, nonspecific DNA binding, and protein oligomerization. The mutation studies also demonstrated the remarkable correlation of oligomeric structure of cII protein to its stability within the bacterial host. An Escherichia coli HtpR- strain has been identified that greatly stabilizes these highly unstable cII mutants.

Amino Acid Sequence↗

PubMed1989

Expression vector with two-step control by the cI-pR-Q-p'R-qut-t'R module of coliphage lambda.

A plasmid expression vector (pCEQ3), using temperature-regulated transcription from the p'R promoter of bacteriophage lambda, has been constructed. The vector is derived from pBR327 in which the EcoRI-ClaI fragment of plasmid DNA is replaced with a 2.2-kb DNA module cI857-pR-Q-p'R-qut-t'R, consisting of two regions of the lambda genome. The first region contains the repressor gene cI857 and promoter pR; the second one contains gene Q and the late promoter p'R. When the repressor protein, product of the cI857 gene, becomes temperature-inactivated, it allows the promoter pR to initiate the transcription of the Q gene. The product of the Q gene, in turn, acts as a positive regulator of transcription from promoter p'R. The promoter activity of pR is fully repressed at a low temperature (30 degrees C) and transcription from p'R is terminated in the absence of Q gene product, but the shift of temperature up to 37 degrees C is sufficient to make the transcription from the p'R promoter highly active. Foreign genes can be inserted into the single EcoRI site downstream from the p'R promoter. The resultant constructions express extremely high levels of the cloned gene product in Escherichia coli.

Bacteriophage lambda↗

PubMed1986

Very short patch mismatch repair in phage lambda: repair sites and length of repair tracts.

Five amber mutations in the repressor (cI) gene of bacteriophage lambda recombine anomalously with nearby cI mutations. When any of these markers is used in four-factor crosses, cI+ recombinants that are expected to require three cross-overs occur at high frequencies. These recombinants are attributable to very-short-patch (VSP) repair of specific mismatches in DNA heteroduplexes formed during recombination between the markers flanking cI. The sites of the repair-prone mutations and the lengths of repair tracts have now been determined. Amber mutations subject to VSP repair are C to T transitions in 5'CCATGG, the sequence methylated by the product of gene dcm, and also in the related 5'CAGG or 5'CCAG sequences. Ambers arising in CAG sequences found in other contexts, or in codons other than CAG, were not subject to VSP repair. Repair tracts rarely, if ever, exceed ten nucleotides in length, and can be as short as two nucleotides. A repair-prone mutation does not stimulate recombination between flanking cI markers.

Bacteriophage lambda↗

PubMed1999

Roles of RuvC and RecG in phage lambda red-mediated recombination.

The recombination properties of Escherichia coli strains expressing the red genes of bacteriophage lambda and lacking recBCD function either by mutation or by expression of lambda gam were examined. The substrates for recombination were nonreplicating lambda chromosomes, introduced by infection; Red-mediated recombination was initiated by a double-strand break created by the action of a restriction endonuclease in the infected cell. In one type of experiment, two phages marked with restriction site polymorphisms were crossed. Efficient formation of recombinant DNA molecules was observed in ruvC+ recG+, ruvC recG+, ruvC+ recG, and ruvC recG hosts. In a second type of experiment, a 1-kb nonhomology was inserted between the double-strand break and the donor chromosome's restriction site marker. In this case, recombinant formation was found to be partially dependent upon ruvC function, especially in a recG mutant background. In a third type of experiment, the recombining partners were the host cell chromosome and a 4-kb linear DNA fragment containing the cat gene, with flanking lac sequences, released from the infecting phage chromosome by restriction enzyme cleavage in the cell; the formation of chloramphenicol-resistant bacterial progeny was measured. Dependence on RuvC varied considerably among the three types of cross. However, in all cases, the frequency of Red-mediated recombination was higher in recG than in recG+. These observations favor models in which RecG tends to push invading 3'-ended strands back out of recombination intermediates.

Bacterial Proteins↗

PubMed2000

Novel fold and capsid-binding properties of the lambda-phage display platform protein gpD.

The crystal structure of gpD, the capsid-stabilizing protein of bacteriophage lambda, was solved at 1.1 A resolution. Data were obtained from twinned crystals in space group P21 and refined with anisotropic temperature factors to an R-factor of 0.098 (Rfree = 0. 132). GpD (109 residues) has a novel fold with an unusually low content of regular secondary structure. Noncrystallographic trimers with substantial intersubunit interfaces were observed. The C-termini are well ordered and located on one side of the trimer, relatively far from its three-fold axis. The N-termini are disordered up to Ser 15, which is close to the three-fold axis and on the same side as the C-termini. A density map of the icosahedral viral capsid at 15 A resolution, obtained by cryo-electron microscopy and image reconstruction, reveals gpD trimers, seemingly indistinguishable from the ones seen in the crystals, at all three-fold sites. The map further reveals that the side of the trimer that binds to the capsid is the side on which both termini reside. Despite this orientation of the gpD trimer, fusion proteins connected by linker peptides to either terminus bind to the capsid, allowing protein and peptide display.

Amino Acid Sequence↗

PubMed1995

Expression and functional characterization of Escherichia coli NusA and lambda Q as glutathione S-transferase fusion proteins.

The Escherichia coli transcription factor NusA and the bacteriophage lambda antiterminator Q proteins were expressed as inducible glutathione S-transferase (GST) fusion proteins. The fusion proteins were purified under nondenaturing conditions by affinity chromatography on glutathione agarose. Thrombin cleavage of the glutathione agarose-bound fusion proteins yielded homogeneously pure NusAN+15 (5 mg/g cells) and almost homogeneously pure QN+13 protein (0.7 mg/g cells), where N+x indicates the presence of x additional amino acids at the N-terminus of the protein. The purified NusAN+15 exhibited the same activities as wildtype NusA in enhancement of transcriptional pausing, enhancement of termination at Rho-independent terminators, and enhancement of Q-mediated antitermination in vitro. The QN+13 protein exhibited both anti-pausing and antitermination activities in Q-mediated transcription antitermination. However, the antitermination activity of QN+13 was lost gradually during storage if the thrombin used for cleavage of the GST fusion protein was not removed. This was due to cleavage by thrombin after Arg22 within the Q protein itself, at a noncanonical thrombin cleavage site, so the truncated protein (QN+22) lacked the first 22 amino acids at the N-terminus of Q. The expression vectors described here can be used to rapidly produce large quantities of these proteins, and the truncated Q protein can be used to evaluate the requirement for the N-terminus of Q in antitermination, anti-pausing, interactions with the DNA template (qut site), and interaction with RNA polymerase itself.

Bacterial Proteins↗

PubMed1985

1H NMR studies of lambda cro repressor. 2. Sequential resonance assignments of the 1H NMR spectrum.

The cro repressor protein from bacteriophage lambda has been studied in solution by two-dimensional nuclear magnetic resonance spectroscopy (2D NMR). Following the approach of Wüthrich and co-workers [Wüthrich, K., Wider, G., Wagner, G., & Braun, W. (1982) J. Mol. Biol. 155, 311-319], individual spin systems were identified by J-correlated spectroscopy (COSY) supplemented, where necessary, by relayed coherence transfer spectroscopy (RELAY). Nuclear Overhauser effect spectroscopy (NOESY) was used to obtain sequence-specific assignments. From the two-dimensional spectra, the peptide backbone resonances (NH and C alpha H) for 65 of the 66 amino acids were assigned, as well as most of the side chain resonances. The chemical shifts for the assigned protons are reported at 35 degrees C in 10 mM potassium phosphate, pH 6.8, and in 10 mM potassium phosphate, pH 4.6, 0.2 M KCl, and 0.1 mM EDTA. Small shifts were observed for some resonances upon addition of salt, but no major changes in the spectrum were seen, indicating that no global structural change occurs between these ionic strengths. NOE patterns characteristic of alpha-helices, beta-strands, and turns are seen in various regions of the primary sequence. From the location of these regions the secondary structure of cro in solution appears to be virtually identical with the crystal structure [Anderson, W. F., Ohlendorf, D. H., Takeda, Y., & Matthews, B. W. (1981) Nature (London) 290, 754-758]. Missing assignments include the Pro-59 resonances and the peripheral protons of the eight lysine, the three arginine, and three of the five isoleucine residues.

Amino Acid Sequence↗

PubMed1979

Escherichia coli RNA polymerase binding and initiation of transcription on fragments of lambda rifd 18 DNA containing promoters for lambda genes and for rrnB, tufB, rplC,A, rplJ,L, and rpoB,C genes.

Promoters of genes for bacteriophage lambda and for Escherichia coli ribosomal RNA (rrnB), elongation factor Tu (tufB), ribosomal proteins L11 (rplK), L1 (rplA), L10 (rplJ), and L7/L12 (rplL), and RNA polymerase subunits beta (rpoB) and beta' (rpoC) were studied by use of two types of filter binding assays which measured E. coli RNA polymerase binding and initiation of transcription on restriction fragments of lambda rifd 18 DNA. The DNA fragments selectively retained on filters were eluted, concentrated, and analyzed by gel electrophoresis. The binding characteristics of these promotor fragments were qualitatively determined by varying the RNA polymerase, salt, and glycerol concentrations in the polymerase binding assay with HaeIII fragments of lambda rifd 18 DNA. The approximate map locations of these small HaeIII fragments were determined by HaeIII digestion of the larger, previously mapped EcoRI, HindIII, and SmaI restriction fragments of the phage DNA. The base compositions proximal to the 5' ends of mRNA's from promoters on these DNA fragments were elucidated by the polymerase initiation assay, in which the addition of various combinations of nucleoside triphosphates to the reaction allowed RNA polymerase to form high-salt-resistant initiation complexes with some of the known SmaI + EcoRI, EcoRI + HindIII, or HaeIII restriction fragments of lambda rifd 18 DNA. The data obtained by this technique are consistent with the map positions and 5' mRNA base sequences of the known lambda promotors p'R, po, pR and pL. In the main focus of this work, we have determined the approximate map locations and 5' mRNA base compositions of several promoters for known E. coli genes including rrnB, tufB, rplK,A, and rplJ,L. No promoter was detected between rplL and the rpoB,C genes. Thus our data are consistent with the conclusion of Yamamoto and Nomura (1978) that the beta and beta' mRNA is probably cotranscribed from the promoter for rplJ,L. Finally, the approximate map positions and the NTP combinations which initiated transcription of several unknown lambda and E. coli in vitro promoters are reported. The methods reported should prove useful for studying the characteristics of promoters on other cloned DNA regions.

Bacteriophage lambda↗

PubMed1991

Assembly of transcription elongation complexes containing the N protein of phage lambda and the Escherichia coli elongation factors NusA, NusB, NusG, and S10.

The transcription antitermination protein, N, of bacteriophage lambda; the Escherichia coli elongation factors NusA, NusB, ribosomal protein S10, and NusG; and a DNA template containing a lambda nut (N-ututilization) site are necessary and sufficient for the highly cooperative formation in vitro of stable transcription complexes containing all five elongation factors. Mutations in the nut site, NusA, or the beta-subunit of RNA polymerase (RNAP) that impair antitermination in vivo also abolish the assembly of a stable complex containing the antitermination factors in vitro. The effects of RNAP mutations on assembly imply that the antitermination factors assemble on the surface of RNAP. We have shown previously that NusA binds directly to transcribing RNAP (Ka approximately 10(7) M-1); Ka = association constant and we show here that S10 also binds directly and specifically to RNAP with an apparent Ka of 10(6) M-1. These observations led to a model for the ordered assembly of the N-modified transcription complex.

Bacterial Proteins↗

PubMed1989

Lambda repressor mutants that are better substrates for RecA-mediated cleavage.

RecA-mediated cleavage of the bacteriophage lambda repressor results in inactivation of the protein and leads to induction of the lambda prophage. Here, we report the identification of three mutations in lambda repressor that significantly increase the rate of RecA-mediated cleavage. These mutations were isolated as intragenic second-site suppressors of a mutation (ind-) which prevents cleavage. Purified repressor proteins that contain both the ind- mutation and one of the second-site mutations undergo cleavage at near wild-type rates. Purified repressors that contain the second-site mutations in otherwise wild-type backgrounds undergo RecA-mediated cleavage at significantly faster rates than wild-type, and form dimers more poorly than the wild-type protein. In related experiments, we found that other repressor mutants that dimerize poorly are also better substrates for RecA-mediated cleavage. Conversely, we show that a covalent disulfide-bonded repressor dimer is resistant to cleavage. These results support a model in which repressor monomers are the only substrate in the cleavage reaction.

Bacteriophage lambda↗

PubMed1982

Quantitative model for gene regulation by lambda phage repressor.

A statistical thermodynamic model has been developed to account for the cooperative interactions of the bacteriophage lambda repressor with the lambda right operator. The model incorporates a general theory for quantitatively interpreting cooperative site-specific equilibrium binding data. Values for all interaction parameters of the model have been evaluated at 37 degrees C, 0.2 M KCl, from results of DNase protection experiments in vitro [A. D. Johnson, B. J. Meyer, & M. Ptashne, Proc. Natl. Acad. Sci. USA (1979) 76, 5061-5065]. With these values, the model predicts repression curves at the divergent promoters PR and PRM that control transcription of genes coding for the regulatory proteins cro and repressor, respectively. At physiological repressor concentrations, repression at PR is predicted to be nearly complete whereas PRM is predicted to remain highly active. The results demonstrate the importance of cooperative interactions between repressor dimers bound to the adjacent operator sites OR1 and OR2 in maintaining a stable lysogenic state and in allowing efficient switchover to the lytic state during induction.

Bacteriophage lambda↗

PubMed1990

A short DNA sequence from lambda phage inhibits protein synthesis in Escherichia coli rap.

The Escherichia coli rap mutant inhibits vegetative growth of bacteriophage lambda. Phage mutations termed bar, which overcome the rap defect, have been mapped to three genetic loci in the pL operon. Plasmids with a lambda wild-type bar DNA segment cloned downstream from an active promoter cannot be maintained in rap mutant bacteria. The viability of a rap mutant strain decreases rapidly after induction of transcription through bar regions present on plasmids. Under these (restrictive) conditions the expression of plasmid-encoded beta-lactamase and plasmid DNA replication are arrested, but plasmid RNA synthesis continues for several hours. Analysis of protein extracts from E. coli rap cells containing bar plasmids revealed that both plasmid and bacterial protein synthesis are inhibited under restrictive conditions. In addition, unlike other RNAs tested, the chemical half-life of bar RNA increases 3.5-fold relative to the half-life of bar RNA under permissive conditions. We propose that transcription through the bar region, or the accumulation of bar RNA, results in an irreversible defect in cellular mRNA translation. This defect eventually kills the rap cells, and thus prevents bar plasmid maintenance.

Bacterial Proteins↗

PubMed2006

Architecture of the 99 bp DNA-six-protein regulatory complex of the lambda att site.

The highly directional and tightly regulated recombination reaction used to site-specifically excise the bacteriophage lambda chromosome out of its E. coli host chromosome requires the binding of six sequence-specific proteins to a 99 bp segment of the phage att site. To gain structural insights into this recombination pathway, we measured 27 FRET distances between eight points on the 99 bp regulatory DNA bound with all six proteins. Triangulation of these distances using a metric matrix distance-geometry algorithm provided coordinates for these eight points. The resulting path for the protein-bound regulatory DNA, which fits well with the genetics, biochemistry, and X-ray crystal structures describing the individual proteins and their interactions with DNA, provides a new structural perspective into the molecular mechanism and regulation of the recombination reaction and illustrates a design by which different families of higher-order complexes can be assembled from different numbers and combinations of the same few proteins.

Algorithms↗

PubMed1999

Kinetic characterization of the GTPase activity of phage lambda terminase: evidence for communication between the two "NTPase" catalytic sites of the enzyme.

The terminase enzyme from bacteriophage lambda is responsible for the insertion of viral DNA into the confined space within the capsid. The enzyme is composed of the virally encoded proteins gpA (73.3 kDa) and gpNu1 (20.4 kDa) isolated as a gpA(1).gpNu1(2) holoenzyme complex. Lambda terminase possesses a site-specific nuclease activity, an ATP-dependent DNA strand-separation activity, and an ATPase activity that must work in concert to effect genome packaging. We have previously characterized the ATPase activity of the holoenzyme and have identified catalytic active sites in each enzyme subunit [Tomka and Catalano (1993) Biochemistry 32, 11992-11997; Hwang et al. (1996) Biochemistry 35, 2796-2803]. We have noted that GTP stimulates the ATPase activity of the enzyme, and terminase-mediated GTP hydrolysis has been observed. The studies presented here describe a kinetic analysis of the GTPase activity of lambda terminase. GTP hydrolysis by the enzyme requires divalent metal, is optimal at alkaline pH, and is strongly inhibited by salt. Interestingly, while GTP can bind to the enzyme in the absence of DNA, GTP hydrolysis is strictly dependent on the presence of polynucleotide. Unlike ATP hydrolysis that occurs at both subunits of the holoenzyme, a single catalytic site is observed in the steady-state kinetic analysis of GTPase activity (k(cat) approximately 37 min(-)(1); K(m) approximately 500 microM). Moreover, while GTP stimulates ATP hydrolysis (apparent K(D) approximately 135 microM for GTP binding), all of the adenosine nucleotides examined strongly inhibit the GTPase activity of the enzyme. The data presented here suggest that the two "NTPase" catalytic sites in terminase holoenzyme communicate, and we propose a model describing allosteric interactions between the two sites. The biological significance of this interaction with respect to the assembly and disassembly of the multiple nucleoprotein packaging complexes required for virus assembly is discussed.

Acid Anhydride Hydrolases↗

PubMed2002

Arm-site binding by lambda -integrase: solution structure and functional characterization of its amino-terminal domain.

The integrase protein (Int) from bacteriophage lambda catalyzes the insertion and excision of the viral genome into and out of Escherichia coli. It is a member of the lambda-Int family of site-specific recombinases that catalyze a diverse array of DNA rearrangements in archaebacteria, eubacteria, and yeast and belongs to the subset of this family that possesses two autonomous DNA-binding domains. The heterobivalent properties of Int can be decomposed into a carboxyl-terminal domain that executes the DNA cleavage and ligation reactions and a smaller amino-terminal domain that binds to an array of conserved DNA sites within the phage arms, thereby arranging Int protomers within the higher-order recombinogenic complex. We have determined that residues Met-1 to Leu-64 of Int constitute the minimal arm-type DNA-binding domain (INT-DBD(1-64)) and solved the solution structure by using NMR. We show that the INT-DBD(1-64) is a novel member of the growing family of three-stranded beta-sheet DNA-binding proteins, because it supplements this motif with a disordered amino-terminal basic tail that is important for arm-site binding. A model of the arm-DNA-binding domain recognizing its cognate DNA site is proposed on the basis of similarities with the analogous domain of Tn916 Int and is discussed in relation to other features of the protein.

Arginine↗

PubMed1998

Crystal structure of lambda-Cro bound to a consensus operator at 3.0 A resolution.

The structure of the Cro protein from bacteriophage lambda in complex with a 19 base-pair DNA duplex that includes the 17 base-pair consensus operator has been determined at 3.0 A resolution. The structure confirms the large changes in the protein and DNA seen previously in a crystallographically distinct low-resolution structure of the complex and, for the first time, reveals the detailed interactions between the side-chains of the protein and the base-pairs of the operator. Relative to the crystal structure of the free protein, the subunits of Cro rotate 53 degrees with respect to each other on binding DNA. At the same time the DNA is bent by 40 degrees through the 19 base-pairs. The intersubunit connection includes a region within the protein core that is structurally reminiscent of the "ball and socket" motif seen in the immunoglobulins and T-cell receptors. The crystal structure of the Cro complex is consistent with virtually all available biochemical and related data. Some of the interactions between Cro and DNA proposed on the basis of model-building are now seen to be correct, but many are different. Tests of the original model by mutagenesis and biochemical analysis corrected some but not all of the errors. Within the limitations of the crystallographic resolution it appears that operator recognition is achieved almost entirely by direct hydrogen-bonding and van der Waals contacts between the protein and the exposed bases within the major groove of the DNA. The discrimination of Cro between the operators OR3 and OR1, which differ in sequence at just three positions, is inferred to result from a combination of small differences, both favorable and unfavorable. A van der Waals contact at one of the positions is of primary importance, while the other two provide smaller, indirect effects. Direct hydrogen bonding is not utilized in this distinction.

Bacteriophage lambda↗

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