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PubMed2001

The solution structure of bacteriophage lambda protein W, a small morphogenetic protein possessing a novel fold.

Protein W (gpW) from bacteriophage lambda is required for the stabilization of DNA within the phage head and for attachment of tails onto the head during morphogenesis. Although comprised of only 68 residues, it likely interacts with at least two other proteins in the mature phage and with DNA. Thus, gpW is an intriguing subject for detailed structural studies. We have determined its solution structure using NMR spectroscopy and have found it to possesses a novel fold consisting of two alpha-helices and a single two-stranded beta-sheet arranged around a well-packed hydrophobic core. The 14 C-terminal residues of gpW, which are essential for function, are unstructured in solution.

Amino Acid Sequence↗

PubMed1980

On the role of the dnaB protein of Escherichia coli in the replication of lambda bacteriophage DNA.

The interaction between the dnaB protein of E. coli and the gene P product of lambda bacteriophage was investigated by measuring the cleavage of closed circular phage DNA after infection of two temperature sensitive dnaB mutants, JG28 and To534 groP- B. Cleavage of superhelical DNA from a lambda pi B mutant phage was observed after infection of either strain whereas superhelical DNA from a wild type phage was only cleaved after infection of JG28. When DNA synthesis in infected cells was blocked by incubation at the nonpermissive temperature, no inhibition of superhelical phage DNA cleavage was observed. It is concluded that in conditions where the dnaB protein has lost the capacity to function in nucleotide polymerization, it is capable of interacting with the lambda replication gene products to introduce a break in the phage DNA.

Bacterial Proteins↗

PubMed1983

Rho-dependent termination of transcription. I. Identification and characterization of termination sites for transcription from the bacteriophage lambda PR promoter.

We have conducted a detailed investigation of in vitro transcription from the bacteriophage lambda PR promoter in order to examine various aspects of the mechanism of rho-dependent termination. In these studies, we have focused particularly on nucleotide sequence specificity, both at the termini and at potential rho-binding sites on the mRNA, and on the relationships between elongation, pausing, and termination. Rho-terminated transcripts from restriction fragment templates have been analyzed by polyacrylamide gel electrophoresis, and termination efficiencies have been established by densitometry of autoradiographs. Termination sites on the template have been located by comparing the electrophoretic mobilities of terminated transcripts with those of transcripts of known length that have been artificially terminated by the incorporation of 3'-O-methyl nucleotides. We have identified five discrete rho-dependent termination sites located between 290 and 450 base pairs downstream from the lambda PR promoter. These rho-dependent 3'-termini are somewhat heterogeneous in details of sequence and potential RNA secondary structure, but all possess features that appear to be characteristic of RNA polymerase elongation pausing sites (Morgan, W. D., Bear, D. G., and von Hippel, P. H. (1983) J. Biol. Chem. 258, 9565-9574). The efficiency of termination at individual sites ranges from 20 to 70% under the usual in vitro transcription conditions; termination is inhibited by increasing the monovalent salt concentration. Lowering nucleoside triphosphate substrate concentrations increases termination efficiency at some sites located 290 or more base pairs downstream from PR, but does not enhance termination at sites closer to PR. The substitution of inosine for guanosine residues in the transcript, which decreases the stability of the RNA-DNA hybrid and of secondary structure in the nascent mRNA, results in strong rho-dependent termination at several new sites located 100 to 260 base pairs downstream from PR. In Morgan et al. (cited above), data on RNA polymerase elongation pausing as a function of reaction conditions are correlated with these termination results, and a general model for rho-dependent termination is discussed.

Bacteriophage lambda↗

PubMed1998

Crystal structure of the lysozyme from bacteriophage lambda and its relationship with V and C-type lysozymes.

Like other lysozymes, the bacteriophage lambda lysozyme is involved in the digestion of bacterial walls. This enzyme is remarkable in that its mechanism of action is different from the classical lysozyme's mechanism. From the point of view of protein evolution, it shows features of lysozymes from different classes. The crystal structure of the enzyme in which all tryptophan residues have been replaced by aza-tryptophan has been solved by X-ray crystallography at 2.3 A using a combination of multiple isomorphous replacement, non-crystallographic symmetry averaging and density modification techniques. There are three molecules in the asymmetric unit. The characteristic structural elements of lysozymes are conserved: each molecule is organized in two domains connected by a helix and the essential catalytic residue (Glu19) is located in the depth of a cleft between the two domains. This cleft shows an open conformation in two of the independent molecules, while access to the cavity is much more restricted in the last one. A structural alignment with T4 lysozyme and hen egg white lysozyme allows us to superpose about 60 C alpha atoms with a rms distance close to 2 A. The best alignments concern the helix preceding the catalytic residue, some parts of the beta sheets and the helix joining the two domains. The results of sequence alignments with the V and C lysozymes, in which weak local similarities had been detected, are compared with the structural results.

Amino Acid Sequence↗

PubMed1996

Bacteriophage lambda N protein alone can induce transcription antitermination in vitro.

Specific and processive antitermination by bacteriophage lambda N protein in vivo and in vitro requires the participation of a large number of Escherichia coli proteins (Nus factors), as well as an RNA hairpin (boxB) within the nut site of the nascent transcript. In this study we show that efficient, though nonprocessive, antitermination can be induced by large concentrations of N alone, even in the absence of a nut site. By adding back individual components of the system, we also show that N with nut+ nascent RNA is much more effective in antitermination than is N alone. This effect is abolished if N is competed away from the nut+ RNA by adding, in trans, an excess of boxB RNA. The addition of NusA makes antitermination by the N-nut+ complex yet more effective. This NusA-dependent increase in antitermination is lost when delta nut transcripts are used. These results suggest the formation of a specific boxB RNA-N-NusA complex within the transcription complex. By assuming an equilibrium model, we estimate a binding constant of 5 x 10(6) M-1 for the interaction of N alone with the transcription complex. This value can be used to estimate a characteristic dissociation time of N from the complex that is comparable to the dwell time of the complex at an average template position, thus explaining the nonprocessivity of the antitermination effect induced by N alone. On this basis, the effective dissociation rate of N should be approximately 1000-fold slower from the minimally processive (100-600 bp) N-NusA-nut+ transcription complex and approximately 10(5)-fold slower from the maximally processive (thousands of base pairs) complex containing all of the components of the in vivo N-dependent antitermination system.

Bacterial Proteins↗

PubMed1996

The Rz1 gene product of bacteriophage lambda is a lipoprotein localized in the outer membrane of Escherichia coli.

The Rz1 gene of bacteriophage lambda is located within the Rz1 lysis gene. It codes for the 6.5-kDa prolipoprotein (Rz1) which undergoes N-terminal signal sequence cleavage and post-translational lipid modification of the N-terminal Cys of the mature protein. Globomycin, the antibiotic which inhibits bacterial signal peptidase II, specific for prolipoproteins containing diacylglyceryl cysteine [Hayashi and Wu, J. Bioenerg. Biomembr. 22 (1990) 451-471] inhibits the N-terminal sequence cleavage of the Rz1 precursor. The mature protein is rich in Pro, which constitutes 25% of its amino acids (aa). Using a computer-predicted, synthetic, 15-aa antigenic determinant of Rz1 polyclonal anti-Rz[46-60] antibodies, were obtained, and employed to localize Rz1 in bacterial fractions. In induced Escherichia coli lambda lysogens Rz1 was found almost exclusively in the outer membrane (OM). In a strain overproducing Rz1 from the pSB54 plasmid, it was distributed in all the fractions, OM, fraction A and inner membrane (IM). Expression of Rz1 from the pSB54 caused enlargement of fraction A, corresponding to the adhesion sites of OM and IM. Such an enlargement was previously observed in induced lambda lysogens, shortly before the onset of lysis.

Amino Acid Sequence↗

PubMed1984

A functional domain of bacteriophage lambda terminase for prohead binding.

Terminase is a multifunctional protein complex involved in DNA packaging during bacteriophage lambda assembly. Terminase is made of gpNul and gpA, the products of the phage lambda Nu1 and A genes. Early during DNA packaging terminase binds to lambda DNA to form a complex called complex I. Terminase is required for the binding of proheads by complex I to form a DNA: terminase: prohead complex known as complex II. Terminase remains associated with the DNA during encapsidation. The other known role for terminase in packaging is the production of staggered nicks in the DNA thereby generating the cohesive ends. Lambdoid phage 21 has cohesive ends identical to those of lambda. The head genes of lambda and 21 show partial sequence homology and are analogous in structure, function and position. The terminases of lambda and 21 are not interchangeable. At least two actions of terminase are involved in this specificity: (1) DNA binding; (2) prohead binding. The 1 and 2 genes at the left end of the 21 chromosome were identified as coding for the 21 terminase. gp1 and gp2 are analogous to gpNu1 and gpA, respectively. We have isolated a phage, lambda-21 hybrid 33, which is the product of a crossover between lambda and 21 within the terminase genes. Lambda-21 hybrid 33 DNA and terminase have phage 21 packaging specificity, as determined by complementation and helper packaging studies. The terminase of lambda-21 hybrid 33 requires lambda proheads for packaging. We have determined the position at which the crossover between lambda DNA and 21 DNA occurred to produce the hybrid phage. Lambda-21 hybrid 33 carries the phage 21 1 gene and a hybrid phage 2/A gene. Sequencing of lambda-21 hybrid 33 DNA shows that it encodes a protein that is homologous at the carboxy terminus with the 38 amino acids of the carboxy terminus of lambda gpA; the remainder of the protein is homologous to gp2. The results of these studies define a specificity domain for prohead binding at the carboxy terminus of gpA.

Bacteriophage lambda↗

PubMed1982

Nucleotide sequence analysis of in vivo recombinants between bacteriophage lambda DNA and pBR322.

The nucleotide sequences involved in the illegitimate recombination of four recombinants between bacteriophage lambda DNA and pBR322 in E. coli (lambda TA6, lambda KA3, lambda TA1R, and lambda KA7) were determined. Each resulted from recombination between regions of homology of 10 to 13 base pairs. The presence of a recA+ allele was found to stimulate recombination between lambda DNA and pBR322 approximately 10-fold. Lambda TA6, lambda KA3, and lambda KA7 were isolated in the presence of a recA+ allele and therefore, may have been generated by the recA recombination system. However, lambda TA1R was isolated in a recA mutant, and was presumably generated by a different recombination system. The possibility that it was generated by DNA gyrase is discussed. Two recombination events were required to form lambda KA7, which may indicate that it also was generated by DNA gyrase.

Alleles↗

PubMed1993

Structural transitions during maturation of bacteriophage lambda capsids.

The three-dimensional structures of the procapsid and of the mature capsid of bacteriophage lambda were determined to a resolution of approximately 3.4 nm by cryo-electron microscopy and image processing. The mature lambda capsid contains two major proteins, gpE and gpD, arranged on a T = 7 lattice, with gpE arranged as hexamers and pentamers and gpD arranged as trimers. The hexamers and pentamers in the virion display a cartwheel-like structure, with skewed spokes (or arms) radiating out from a central hexameric hub. The thimble-shaped gpD trimers are superimposed on the trivalent interaction point of these arms. A reconstruction of a lambda D- mutant capsid to lower resolution shows no trace of these trimers, thus revealing the interactions of the underlying arms. The procapsid has elongated, irregularly shaped hexamers with gpE subunits set perpendicularly to the capsid surface.

Bacteriophage lambda↗

PubMed2001

Bacteriophage lambda: alive and well and still doing its thing.

The lambda (lambda) family of bacteriophages continues to provide significant insights into the understanding of basic biological processes, as well as useful technological innovations. Areas in which recent advances have occurred include transcription elongation, repressor interactions, genomics and post-transcriptional regulation. The homologous lambda recombination functions have been exploited as an efficient in vivo recombinant engineering system for functional genomic studies. The virulence of some pathogenic strains of Escherichia coli is enhanced by the expression of Shiga toxin (stx) genes encoded on a resident lambdoid prophage. Recent work suggests that the phage regulatory network may be a significant contributor to toxin production and release by these pathogenic E. coli.

Bacteriophage lambda↗

PubMed2000

The C-terminal portion of the tail fiber protein of bacteriophage lambda is responsible for binding to LamB, its receptor at the surface of Escherichia coli K-12.

Bacteriophage lambda adsorbs to its Escherichia coli K-12 host by interacting with LamB, its cell-surface receptor. We fused C-terminal portions of J, the tail fiber protein of lambda, to maltose-binding protein. Solid-phase binding assays demonstrated that a purified fusion protein comprising only the last 249 residues of J could bind to LamB trimers and inhibited recognition by anti-LamB antibodies. Electron microscopy further demonstrated that the fusion protein could also bind to LamB at the surface of intact cells. This interaction prevented lambda adsorption but affected only partially maltose uptake.

ATP-Binding Cassette Transporters↗

PubMed1986

Specialized nucleoprotein structures at the origin of replication of bacteriophage lambda: localized unwinding of duplex DNA by a six-protein reaction.

The O protein of bacteriophage lambda localizes the initiation of DNA replication to a unique site on the lambda genome, ori lambda. By means of electron microscopy, we infer that the binding of O to ori lambda initiates a series of protein addition and transfer reactions that culminate in localized unwinding of the origin DNA, generating a prepriming structure for the initiation of DNA replication. We can define three stages of this prepriming reaction, the first two of which we have characterized previously. First, dimeric O protein binds to multiple DNA binding sites and self-associates to form a nucleoprotein structure, the O-some. Second, lambda P and host DnaB proteins interact with the O-some to generate a larger complex that includes additional DNA from an A + T-rich region adjacent to the O binding sites. Third, the addition of the DnaJ, DnaK, and Ssb proteins and ATP results in an origin-specific unwinding reaction, probably catalyzed by the helicase activity of DnaB. The unwinding reaction is unidirectional, proceeding "rightward" from the origin. The minimal DNA sequence competent for unwinding consists of two O binding sites and the adjacent A + T-rich region to the right of the binding sites. We conclude that the lambda O protein localizes and initiates a six-protein sequential reaction responsible for but preceding the precise initiation of DNA replication. Specialized nucleoprotein structures similar to the O-some may be a general feature of DNA transactions requiring extraordinary precision in localization and control.

Adenosine Triphosphate↗

PubMed1985

Interaction of rho factor with bacteriophage lambda cro gene transcripts.

Rho protein is responsible for termination of transcription of the cro gene of bacteriophage lambda. Since rho is known to interact with the RNA whose synthesis is being terminated, we measured the specificity and strength of binding of rho to isolated cro transcripts, using a nitrocellulose filter retention assay. The association constant (K alpha) for the binding of rho to a 372-nucleotide cro transcript was determined to be 7 +/- 2 X 10(8) M-1 at 37 degrees C and about 20-fold less at 4 degrees C. Although NTP cleavage is required for rho activity, the presence of ATP did not alter the K alpha. Rho bound less tightly (K alpha less than 10(8) M-1) to partial cro transcripts smaller than 290 nucleotides and had very little affinity (K alpha less than 10(6) M-1) for lambda 4 S RNA, lambda 6 S RNA, and partial cro transcripts smaller than 160 nucleotides. In contrast, cro transcripts as short as 100 nucleotides bound if guanosine residues were replaced with inosine. In addition, rho bound readily to 3' fragments of cro RNA that had 85 or more residues. A common feature of the RNA molecules that bind tightly to rho protein is that they have a stretch of at least 85 nucleotides with relatively few (less than 14%) guanosine residues. Such a segment is thus likely to be largely single-stranded. These results suggest that the binding of rho to lambda cro mRNA is dependent on a 3' terminal segment that has those properties.

Bacteriophage lambda↗

PubMed1994

A role for residue 151 of LamB in bacteriophage lambda adsorption: possible steric effect of amino acid substitutions.

LamB is the cell surface receptor for bacteriophage lambda. LamB missense mutations yielding resistance to lambda have been previously grouped in two classes. Class I mutants block growth of lambda with wild-type host range (lambda h+) but support growth of one-step extended-host-range mutants (lambda h). Class II mutants block lambda h but support growth of two-step extended host range mutants (lambda hh*). While Class I mutations occur at 11 different amino acid sites, in five distinct portions of LamB, all the Class II mutations analyzed previously correspond to the same G-to-D change at amino acid 151. We generated by in vitro mutagenesis four different new substitutions at site 151 (to S, V, R, and C). Two of the mutants (G-151-->V [G151V] and G151R) were of Class II, while the two others (G151S and G151C) were of Class I, demonstrating that not only the site but also the nature of the substitutions at residue 151 was critical for the phage sensitivity phenotypes. The introduction of a negatively charged, a positively charged, or an aliphatic nonpolar residue at site 151 of LamB prevented both lambda h+ and lambda h adsorption, indicating that the block is not due to a charge effect. In contrast to G151D, which was sensitive to all the lambda hh* phages, G151V and G151R conferred sensitivity to only four of the five lambda hh* phages. Thus, G151V and G151R represent a new subclass of Class II LamB mutations that is more restrictive with respect to the growth of lambda hh*. Our results agree with the hypothesis that residue 151 belongs to an accessibility gate controlling the access to the phage tight-binding site and that substitutions at this residue affect the access of the phage to the binding site in relation to the size of the substitute side chain (surface area): the most restrictive changes are G151V and G151R, followed to a lesser extent by G151D and they by G151S and G151C.

Bacterial Outer Membrane Proteins↗

PubMed1989

Transcriptional activation of bacteriophage lambda DNA replication in vitro: regulatory role of histone-like protein HU of Escherichia coli.

Initiation of bacteriophage lambda DNA replication in vivo and in crude in vitro systems is strongly dependent on transcription at or near the lambda replication origin (ori lambda). Through its capacity to prevent RNA polymerase-mediated 'transcriptional activation' of lambda DNA replication, the lambda cI repressor is capable of negatively regulating initiation of lambda DNA replication, even when all required replication proteins are present. Surprisingly, the strict requirement for transcriptional activation of lambda DNA replication was lost when lambda replication was initiated in an in vitro system composed of nine purified replication proteins [Mensa-Wilmot et al. (1989) J. Biol. Chem., 264, 2853-2861]. We have found that crude extracts of Escherichia coli contain proteins that are capable of restoring the physiological linkage between transcription and ori lambda-dependent replication when they are added to the nine-protein replication system. The protein primarily responsible for this effect has been purified and identified as protein HU, a histone-like protein that is a major constituent of the bacterial nucleoid. HU, when present at a 1:1 weight ratio with supercoiled ori lambda plasmid, is a potent inhibitor of lambda DNA replication in the nine-protein replication system. However, when the ori lambda template is transcribed by E. coli RNA polymerase, the HU-mediated inhibition of lambda DNA replication is abolished. HU does not inhibit propagation of lambda replication forks. Instead, HU apparently interferes with the assembly or function of nucleoprotein structures containing the E. coli DnaB helicase that are formed at ori lambda prior to priming and DNA synthesis. We suggest that the chromatin structure of the template DNA in the region surrounding ori lambda plays a central role in the negative regulation of the initiation of lambda DNA replication in vivo.

Bacterial Proteins↗

PubMed1983

Structure and inherent properties of the bacteriophage lambda head shell. III. Spectroscopic studies on the expansion of the prohead.

The head shell of bacteriophage lambda expands by about 20% in diameter when it packages the DNA molecule in vivo. The expansion reaction is essentially a conformational change of the major head protein molecules to a state of lower free energy and can also be triggered in vitro by treatment with 4 M-urea. In order to investigate the conformational change, we have measured the circular dichroism, fluorescence and difference absorption spectra of the lambda head shell before and after the expansion by the treatment with urea. The far-ultraviolet circular dichroism spectra and the fluorescence spectra show that the expansion is not accompanied by a great change in the secondary structure (29% alpha-helix, 23% beta-structure) and the environment (non-polar) of the tryptophan residues of the major head protein molecule. On the other hand, by measurements of the circular dichroism and difference absorption spectra in the near-ultraviolet region as well as by chemical modification experiments with tetranitromethane, we have found that one or two tyrosine residues of the major head protein are transferred from a polar, solvent-exposed to a non-polar, solvent-unexposed environment during the expansion. Judging from these results, the conformational change seems to be mainly intermolecular or interdomainal rather than intradomainal.

Bacteriophage lambda↗

PubMed1989

Heat shock protein-mediated disassembly of nucleoprotein structures is required for the initiation of bacteriophage lambda DNA replication.

Three Escherichia coli heat shock proteins, DnaJ, DnaK, and GrpE, are required for replication of the bacteriophage lambda chromosome in vivo. We show that the GrpE heat shock protein is not required for initiation of lambda DNA replication in vitro when the concentration of DnaK is sufficiently high. GrpE does, however, greatly potentiate the action of DnaK in the initiation process when the DnaK concentration is reduced to a subsaturating level. We demonstrate in the accompanying articles (Alfano, C. and McMacken, R. (1989) J. Biol. Chem. 264, 10699-10708; Dodson, M., McMacken, R., and Echols, H. (1989) J. Biol. Chem. 264, 10719-10725) that DnaJ and DnaK bind to prepriming nucleoprotein structures that are assembled at the lambda replication origin (ori lambda). Binding of DnaJ and DnaK completes the ordered assembly of an ori lambda initiation complex that also contains the lambda O and P initiators and the E. coli DnaB helicase. With the addition of ATP, the DnaJ and DnaK heat shock proteins mediate the partial disassembly of the initiation complex, and the P and DnaJ proteins are largely removed from the template. Concomitantly, on supercoiled ori lambda plasmid templates, the intrinsic helicase activity of DnaB is activated and DnaB initiates localized unwinding of the DNA duplex, thereby preparing the template for priming and DNA chain elongation. We infer from our results that DnaK and DnaJ function in normal E. coli metabolism to promote ATP-dependent protein unfolding and disassembly reactions. We also provide evidence that neither the lambda O and P initiators nor the E. coli DnaJ and DnaK heat shock proteins play a direct role in the propagation of lambda replication forks in vitro.

Bacterial Proteins↗

PubMed2005

Two-stage continuous operation of recombinant Escherichia coli using the bacteriophage lambda Q- vector.

A two-stage continuous culture of Escherichia coli in combination with a bacteriophage lambda system was performed in order to overcome the intrinsic plasmid instability that is frequently observed in recombinant fermentation. A phage lambda vector with a Q(-) mutation was used to enhance the expression of the lambda system. The optimal values of the important operational variables such as the substrate concentration, the dilution rate, and the mean residence time on the expression of the cloned gene were determined in both batch and continuous cultures. For all culturing modes, the full induction of the cloned gene was observed 4 h after the temperature shift. In the two stage continuous culture, the overproduction reached their maxima at D=0.25 h(-1) with 1.5 S(0) of the medium supply. The maximum productivity of the total beta-galactosidase was 16.3x10(6) U l(-1) h(-1), which was approximately seven times higher than that in the single-copy lysogenic stage. The recombinant cells were stable in the lysogenic state for more than 260 h, while they were stable for 40 h in the lytic state. The instability that developed rapidly in the second tank is believed to be due to the accumulation of lysis proteins as a result of vector leakage during the operation.

Bacteriophage lambda↗