Structure and inherent properties of the bacteriophage lambda head shell. V. Amber mutants in gene E

A total of 940 amber mutants in gene E of bacteriophage lambda was isolated to study the structure-function relationship of the gene product, the major capsid protein. The mutants were mapped to 43 mutation sites, most of which have been located, albeit tentatively, at exact points in the known base sequence, by deletion mapping and by the specificity of mutagenesis and the patterns of suppression. The patterns of suppression were interpreted in terms of both the efficiency of insertion of amino acid residues by suppressors and the exchangeability of amino acid residues. The exchangeability seems to be related to the hydrophilicity of the residues themselves and their environment, as well as to the functional similarity between the replaced and the inserted amino acid residues. Suppression of two of the mutations resulted in the production of characteristic aberrant head-related structures, each showing a defect in a different functional site in the protein. This, together with the approximate positions of some specific missense mutations as determined in this study, revealed the distribution of the functional sites along the polypeptide chain of the gene E product.     

Isolation and characterization of prophage mutants of the defective Bacillus subtilis bacteriophage PBSX.

Bacillus subtilis mutants with lesions in PBSX prophage genes have been isolated. One of these appears to be a regulatory mutant and is defective for mitomycin C-induced derepression of PBSX; the others are defective for phage capsid formation. All of the PBSX structural proteins are synthesized during induction of the capsid defective mutants; however, several of these proteins exhibit abnormal serological reactivity with anti-PBSX antiserum. The two head proteins X4 and X7 are not immunoprecipitable in a mutant which fails to assemble phage head structures. In the tail mutant, proteins X5 and X6 are not immunoprecipitable, tails are not assembled, and a possible tail protein precursor remains uncleaved. The noninducible mutant does not synthesize any PBSX structural proteins after exposure to mitomycin C. The mutation is specific for PBSX since ø105 and SPO2 lysogens of the mutant are inducible. All of the known PBSX-specific mutations were shown to be clustered between argC and metC on the host chromosome. In addition, the metC marker was shown to be present in multiple copies in cells induced for PBSX replication. This suggests that the derepressed prophage replicates while still integrated and that replication extends into the adjacent regions of the host chromosome.     

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.     

Structure and inherent properties of the bacteriophage lambda head shell. VII. Molecular design of the form-determining major capsid protein

Some mutations in the major capsid protein (gpE) of lambda phage can alter the size and shape of the head shell or block the pathway of head maturation. Previous studies on the classification of such mutants showed that there are at least five functional sites on the gpE molecule. In this study, we determined the amino acid exchanges by DNA sequencing to elucidate the molecular design of the form-determining multifunctional protein gpE. In addition, we characterized the mutated gpE molecules by two-dimensional gel electrophoresis and studied suppression patterns of amber mutants at 43 amino acid residues. Those mutations map at 19 amino acid residues at 22 bases, which are located in three regions, 40 to 91, 222 to 246, and 284 to 324 of the 341 amino acid residues of gpE. These regions seem to be important in the activity of gpE, since amber mutations in these regions are suppressed on the average by less species of suppressors than those outside these regions. The mutations having different phenotypes are not segregated from each other, while some mutations having the same phenotype are separated far apart in the primary structure. This suggests that the functional sites were formed during evolution after the folding pattern of the ancestral gpE polypeptide chain had been established. Many of the mutations are located at serine, glycine and proline residues in predicted beta-turns.     

Characterization of bacteriophage lambda excisionase mutants defective in DNA binding

The bacteriophage lambda excisionase (Xis) is a sequence-specific DNA binding protein required for excisive recombination. Xis binds cooperatively to two DNA sites arranged as direct repeats on the phage DNA. Efficient excision is achieved through a cooperative interaction between Xis and the host-encoded factor for inversion stimulation as well as a cooperative interaction between Xis and integrase. The secondary structure of the Xis protein was predicted to contain a typical amphipathic helix that spans residues 18 to 28. Several mutants, defective in promoting excision in vivo, were isolated with mutations at positions encoding polar amino acids in the putative helix (T. E. Numrych, R. I. Gumport, and J. F. Gardner, EMBO J. 11:3797-3806, 1992). We substituted alanines for the polar amino acids in this region. Mutant proteins with substitutions for polar amino acids in the amino-terminal region of the putative helix exhibited decreased excision in vivo and were defective in DNA binding. In addition, an alanine substitution at glutamic acid 40 also resulted in altered DNA binding. This indicates that the hydrophilic face of the alpha-helix and the region containing glutamic acid 40 may form the DNA binding surfaces of the Xis protein.     

Spontaneous lambda OR mutations suppress inhibition of bacteriophage growth by nonimmune exclusion phenotype of defective lambda prophage.

Survivor clones with defects in gene functions that participate in the replicative killing of thermally induced Escherichia coli constructs with integrated lambda N through P or cIII through P gene fragments were selected at a frequency of about 10(-6). Among the population of survivors, clones were identified that exhibited normal lambda immunity at 30 degrees C, as shown by their ability to prevent the plating of lambda wild type and to support the plating of a nearly identical heteroimmune bacteriophage lambda imm434. However, when placed at 42 degrees C to inactivate the cIts857 repressor, these survivor isolates excluded the plating of both lambda wild-type and lambda imm434 phages, a phenotype designated nonimmune exclusion (Nie). Spontaneous mutants of lambda wild type were isolated that overcame the Nie phenotype and would plaque at 42 degrees C on cell lawns of these isolates. The acquired lambda se mutations suppressed nonimmune exclusion, prevented lysogenization by interrupting repressor expression from PRM, and made the phage insensitive to replicative inhibition. The se mutations were genetically mapped and sequenced within the rightward lambda operator site.     

Bacillus subtilis mutants defective in bacteriophage phi 29 head assembly.

Virus assembly mutants of asporogenous Bacillus subtilis defective in bacteriophage phi 29 head assembly were detected by the use of antibodies that reacted strongly with the free dodecameric phi 29 portal vertex composed of gene product 10 (gp10) but weakly with the portal vertex assembled into proheads or phage. Phage adsorption and the synthesis of phage proteins, DNA-gene product 3, and prohead RNA were normal in these mutants, but prohead and phage production was greatly reduced. The assembly defect was transferred to competent B. subtilis by transformation and transduction. PBS1 transduction showed that the vam locus was linked to Tn917 located at 317 degrees on the B. subtilis chromosome.     

Isolation and characterization of specialized lambda transducing bacteriophage carrying the metBJF methionine gene cluster.

Secondary attachment site lysogens of Deltaatt(lambda)Deltappc-argECBH strains of Escherichia coli with lambdacI857 integrated into the bfe gene (88 min) were isolated. Of 20 such lysogens examined, 2 produce lysates with transducing phage containing the metBJF gene cluster (87 min). Reintroduction of the ppc-argECBH chromosome segment (which lies between the bfe and met genes) into these strains virtually abolishes the production of met transducing phage. All of the phage examined have lost essential genes from the left arm of the lambda chromosome. Approximately 85% of the phage appear to have the same genetic composition, containing the metBJF gene cluster, but not the closely linked gene cytR, and having lost phage genes G and J. Analytical CsCl density gradient centrifugation of five representatives of this major class of phage shows four of them to have identical densities (lighter than lambda), while the fifth cannot be resolved from lambda. The four apparently identical phage were isolated from three separate lysates, which suggests the existence of preferred sites for illegitimate recombination on the bacterial and phage chromosomes. Three specialized transducing phage that carry cytR in addition to metB, metJ, and metF have also been studied. Each of these viruses has a different amount of phage deoxyribonucleic acid. Two of them have less deoxyribonucleic acid than lambda, whereas the third has about the same amount. The metB, metF, and cytR genes of the transducing phage have been shown to function in vivo. The phage-borne metB and metF genes are subject to metJ-mediated repression.     

Isolation and characterization of mutations in the bacteriophage lambda terminase genes.

The terminase enzyme of bacteriophage lambda is a hetero-oligomeric protein which catalyzes the site-specific endonucleolytic cleavage of lambda DNA and its packaging into phage proheads; it is composed of the products of the lambda Nul and A genes. We have developed a simple method to select mutations in the terminase genes carried on a high-copy-number plasmid, based on the ability of wild-type terminase to kill recA strains of Escherichia coli. Sixty-three different spontaneous mutations and 13 linker insertion mutations were isolated by this method and analyzed. Extracts of cells transformed by mutant plasmids displayed variable degrees of reduction in the activity of one or both terminase subunits as assayed by in vitro lambda DNA packaging. A method of genetically mapping plasmid-borne mutations in the A gene by measuring their ability to rescue various lambda Aam phages showed that the A mutations were fairly evenly distributed across the gene. Mutant A genes were also subcloned into overproducing plasmid constructs, and it was determined that more than half of them directed the synthesis of normal amounts of full-length A protein. Three of the A gene mutants displayed dramatically reduced in vitro packaging activity only when immature (uncut) lambda DNA was used as the substrate; therefore, these mutations may lie in the endonuclease domain of terminase. Interestingly, the putative endonuclease mutations mapped in two distinct locations in the A gene separated by a least 400 bp.     

Isolation and characterization of novel F-plasmid sopB mutants defective in gene silencing

The product of the sopB gene on the Escherichia coli F-plasmid has been shown to silence genes in the vicinity of its binding region, sopC, when overexpressed. We searched for mutants defective in SopB-dependent silencing by screening for a plasmid incompatibility phenotype, in order to examine the relationship between gene silencing and the intracellular localization of SopB, as revealed by a green fluorescent protein (GFP)-SopB fusion. Nine new mutants were isolated. One of them, in which leucine 92 is replaced by proline, was completely compatible with a sopC-carrying plasmid and was defective in other silencing activities. When expressed as a GFP fusion protein, the L92P mutant was found to be uniformly distributed in the cell. This implies a link between silencing and SopB localization, supporting the view that a high local concentration of SopB drives non-specific DNA binding in segments of the plasmid adjacent to sopC. Despite the lack of apparent localization of GFP fluorescence, the mutant protein, like the wild-type SopB, was found mostly in the inner membrane fraction, indicating that the association with the inner membrane was retained.     

Properties of a mutant of Escherichia coli defective in bacteriophage lambda head formation (groE). II. The propagation of phage lambda

In the accompanying paper (Sternberg, 1973) the properties of three independently isolated strains of Escherichia coli with groE mutations (NS-1, NS-2 and NS-3) have been characterized. In this report the ability of these strains to propagate phage λ is examined in greater detail. In the temperature -sensitive groE strain NS-1, all early phage functions tested (curing, infective center formation, DNA synthesis and early messenger RNA synthesis) are expressed normally. In addition, two late phage functions (late mRNA synthesis and tail formation) are also expressed normally, and a third, phage-induced cell lysis, is expressed with only a slight delay. Based upon head-tail in vitro complementation assays, however, λ fails to make any functional heads at elevated temperatures (41 °C) in this host. Electron microscopic studies of strain NS-1 defective lysates indicate that aberrant head-like forms, including tubular forms and “monsters,” are made.Mutants of λ, designated λEP, which are able to grow in the three groE strains, have been isolated. An analysis of these mutants indicates that at least some carry a mutation in λ head gene E and these make reduced levels of active gene E protein in groE hosts.A further study of all known λ head genes indicates that it is the interaction between the gene E protein and the proteins specified by head genes B and C that is adversely affected by the groE mutation. Presumably, the relative level of gene E protein is too high in groE strains for proper head formation. The λEP mutation compensates for this effect by reducing the level of this protein, and so restoring a balance.     

Structure and function of mutants in the P gene of bacteriophage lambda leading to the pi phenotype

The location of 14 independently isolated spontaneous pi A and pi B point mutants in the lambda P gene and their base exchanges were determined. It was found that the pi B mutation is one unique type mapping close to other pi A mutants. The number of possible pi A mutation sites could be estimated. The mutation sites are distributed asymmetrically in the gene. The N-terminal half of the protein is unchanged. It is assumed to be required for the interaction with the lambda O protein. The P protein can be changed by substitution of a limited number of amino acids at the C-terminus. All functional proteins of this type have pi character. pi proteins do not appear to have altered intracellular levels or stabilities as compared to wild-type P protein. The plating characteristics of our mutants on two groP- mutants located in the dnaJ and dnaK genes, respectively, are strikingly different.     

Isolation and characterization ofAcinetobacter sp. mutants defective in exopolysaccharide biosynthesis

Nitrosoguanidine-induced mutants ofAcinetobacter sp. defective in exopolysaccharide biosynthesis did not differ from the parent strain in distinguishing physiological and biochemical properties, such as requirements for growth factors, utilization of mono- and disaccharides, and resistance to antibiotics. The genetic relation of parent and mutant strains was shown by 16S rRNA PCR analysis. The comparative study of parent and mutant strains with respect to resistance to unfavorable environmental factors confirmed our hypothesis thatAcinetobacter sp. exopolysaccharides perform protective functions. Hybridization experiments revealed the conjugal transfer of plasmid R68.45 fromPseudomonas putida BS228 (R68.45) to mutant but not to the parentAcinetobacter sp. strains. The role of theAcinetobacter sp. exopolysaccharides in providing the genetic stability of this bacterium is discussed.     

Properties of a mutant of Escherichia coli defective in bacteriophage lambda head formation (groE). I. Initial characterization

Three mutant strains of Escherichia coli were independently isolated based upon their inability to propagate bacteriophage λ. The strain most extensively studied, NS-1, has a pleiotropic temperature sensitive alteration that affects cell growth, stable RNA synthesis and λ propagation. Labeling experiments and colorimetric determinations of total RNA carried out in this strain demonstrate that within the first five minutes after raising the temperature to 44.5 °C the rate of total RNA accumulation is reduced to a level that is about 15% that of the control, while protein and DNA synthesis continue at nearly normal rates for at least 30 min. This effect is either due to a very rapid degradation of stable RNA species or a reduced synthesis of RNA. Although the accumulation of all stable RNA species (23, 16 and 4 S RNAs) is reduced co-ordinately to levels ranging from 12 to 16% that of the control, the synthesis of messenger RNA is affected to a lesser degree, if at all. The defect in RNA accumulation can be partially reversed by the addition of chloramphenicol at the moment of temperature shift.In addition to phage λ these strains are unable to propagate RNA phage R17 and lambdoid phages φ80, 21 and 434 at elevated temperatures. The growth of phages T4, T7, P1 and P2 is normal.A genetic analysis of strain NS-1 indicates that all of its temperature sensitive properties depend on a mutation, designated groE-1, which co-transduces with a mel (melibiose) marker. However, the expression of the RNA synthesis defect requires, in addition, a second mutation which does not co-transduce with mel.     

Isolation and characterization of Saccharomyces cerevisiae mutants defective in glycerol catabolism.

Mutants of the yeast Saccharomyces cerevisiae that are defective in the catabolism of glycerol were isolated, and two types of mutants were obtained. One type was deficient in glycerol kinase activity, whereas the other type was deficient in sn-glycerol 3-phosphate dehydrogenase activity. Genetic analysis indicated that each mutant strain owed its phenotype to a single nuclear mutation, and that the two mutations were complementary. The mutations were not linked to each other or to any of 10 loci tested. In addition, neither mutation was centromere linked. Possible mechanisms for the regulation of these enzymes were tested by growing the parental strain in the presence of various carbon sources.     

Isolation and characterization of the host protein groE involved in bacteriophage lambda assembly.

The groE protein, which is involved in the morphogenesis of several bacteriophages, was isolated using a hybrid bacteriophage λ strain which overproduces it. The protein was characterized using biophysical methods, electron microscopy and digital image processing. We postulate that the gp groE complex contains 14 subunits in a cylindrical aggregate with 7-fold rotational symmetry. Possible mechanisms are discussed for the action of this complex in phage morphogenesis.     

Isolation and in vitro characterization of temperature-sensitive mutants of the bacteriophage f1 gene V protein

In vivo selections were used to isolate 43 temperature-sensitive gene V mutants of the bacteriophage f1 from a collection of mutants constructed by saturation mutagenesis of the gene. The sites of temperature-sensitive substitutions are found in both the beta-sheets and the turns of the protein, and some sites are exposed to the solvent while others are not. Thirteen of the variant proteins were purified and characterized to evaluate their free energy changes upon unfolding and their affinities for single-stranded DNA, and eight were tested for their tendencies to aggregate at 42 degrees C. Each of the three temperature-sensitive mutants at buried sites and six of ten at surface sites had free energy changes of unfolding substantially lower (less stabilizing) than the wild-type at 25 degrees C. A seventh mutant at a surface site had a substantially altered unfolding transition and its free energy of unfolding was not estimated. The affinities of the mutant proteins for single-stranded DNA varied considerably, but two mutants at a surface site, Lys69, had much weaker binding to single-stranded DNA than any of the other mutants, while two mutants at another surface site, Glu30, had the highest DNA-binding affinities. The wild-type gene V protein is stable at 42 degrees C, but six of the eight mutants tested aggregated within a few minutes and the remaining two aggregated within 30 minutes at this temperature. Overall, each of the temperature-sensitive proteins tested had a tendency to aggregate at 42 degrees C, and most also had either a low free energy of unfolding (at 25 degrees C), or weak DNA binding. We suggest that any of these properties can lead to a temperature-sensitive gene V phenotype.     

Isolation and genetic characterization of Escherichia coli mutants defective in propionate metabolism.

Escherichia coli mutants defective in propionate metabolism (Prp-) were isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Prp- mutants demonstrate a phenotypic inability to grow on odd-chain-length fatty acids. The new genetic locus for the Prp- phenotype maps at approximately 98 min on the E. coli chromosome.