Host participation in bacteriophage lambda head assembly

Mutants of Escherichia coli, called groE, specifically block assembly of bacteriophage λ heads. When groE bacteria are infected by wild type λ, phage adsorption, DNA injection and replication, tail assembly, and cell lysis are all normal. No active heads are formed, however, and head related “monsters” are seen in lysates. These monsters are similar to the structures seen on infection of wild-type cells by phage defective in genes B or C.We have isolated mutants of λ which can overcome the block in groE hosts and have mapped these mutants. All groE mutations can be compensated for by mutation of phage gene E (hence the name groE). Gene E codes for the major structural subunit of the phage head. Some groE mutants, called groEB, can be compensated by mutation in either gene E or in gene B. Gene B is another head gene.During normal head assembly the protein encoded by phage head gene B or C appears to be converted to a lower molecular weight form, h3, which is found in phage. The appearance of h3 protein in fast sedimenting head related structures requires the host groE function.We suggest that the proteins encoded by phage genes E, B and C, and the bacterial component defined by groE mutations act together at an early stage in head assembly.     

Cleavage of head and tail proteins during bacteriophage T5 assembly: selective host involvement in the cleavage of a tail protein

Electrophoresis studies showed that at least three phage-specified proteins undergo proteolytic cleavage during the development of bacteriophage T5. One of these proteins has a molecular weight of about 135,000 and the product of this cleavage reaction is a minor component of the T5 tail, having a molecular weight of about 128,000. All of the tail-defective T5 mutants studied in this report failed to induce this cleavage reaction under restrictive conditions. This reaction also failed to occur in Escherichia coli groEA639 and groEA36 infected with wild type T5. Examination of lysates of infected groE cells in the electron microscope revealed the presence of filled and empty heads as well as tubular head structures, but no tails were detected. The filled heads were able to combine with separately prepared T5 tails in vitro to form infectious phage particles. Therefore, propagation of T5 in these groE mutants is prevented primarily by a specific block in tail assembly. A T5 mutant, T5?6, was isolated, which has the capacity to propagate in these groE hosts. The gene locus in T5?6 was mapped.The second T5 protein which is cleaved has a molecular weight of 50,000 and is related to head morphogenesis. Treatment of infected cells with l-canavanine (50 μg/ml) inhibited cleavage of this polypeptide. Only small quantities of the major head protein (32,000 mol. wt) were produced in these treated cells. Treatment with canavanine lead to production of tubular heads. The major protein component of partially purified tubular heads has a molecular weight of 50,000. Cells infected with T5 amber H30b, a mutant defective in head gene D20, does not produce the 50,000 and 32,000 molecular weight proteins. These findings suggest that the 50,000 molecular weight protein undergoes cleavage to form the major head polypeptide. A third T5 protein is cleaved to form a minor head component with a molecular weight of 43,000 and its cleavage is linked to that involving the major head protein.     

Mutational Analysis of Mating Type Inheritance in Syngen 4 of PARAMECIUM AURELIA

Six genic mutations restricting clones to mating type VII (O) were isolated in syngen 4, Paramecium aurelia. The only three extensively tested were neither allelic nor closely linked. A second type of mutation, allelic to one of the O restricted mutants, was also found. Clones homozygous for this mutant gene were selfers, producing both O and E (VIII) mating types, but only when they were progeny of mating type E parental clones. While all seven mutant genes behaved as recessives in monohybrid crosses, clones heterozygous at two different loci often demonstrated an unanticipated phenotype: selfing. The significance of the findings is discussed in relation to mating type determination and the evolution of mating type systems.     

Head and tail development of the Drosophila embryo involves spalt, a novel homeotic gene

Mutations in spalt (sal), a novel homeotic gene on the second chromosome of Drosophila, cause opposite transformations in two subterminal regions of the embryo: posterior head segments are transformed into anterior thoracic structures and anterior tail segments are transformed into posterior abdominal structures. The embryonic phenotypes of double mutants for sal and various Antennapedia (ANT-C) or bithorax (BX-C) genes indicate that sal acts independently of the hierarchical order of the latter gene complexes. Trans-regulatory gene mutations causing ectopic expression of ANT-C and BX-C genes do not change the realms of sal action. It is proposed that the region-specific action of the sal gene primarily promotes head as opposed to trunk development, while the BX-C gene AbdB distinguishes tail from head.     

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.     

Identification of Atg2 and ArfGAP1 as Candidate Genetic Modifiers of the Eye Pigmentation Phenotype of Adaptor Protein-3 (AP-3) Mutants in Drosophila melanogaster

The Adaptor Protein (AP)-3 complex is an evolutionary conserved, molecular sorting device that mediates the intracellular trafficking of proteins to lysosomes and related organelles. Genetic defects in AP-3 subunits lead to impaired biogenesis of lysosome-related organelles (LROs) such as mammalian melanosomes and insect eye pigment granules. In this work, we have performed a forward screening for genetic modifiers of AP-3 function in the fruit fly, Drosophila melanogaster. Specifically, we have tested collections of large multi-gene deletions–which together covered most of the autosomal chromosomes–to identify chromosomal regions that, when deleted in single copy, enhanced or ameliorated the eye pigmentation phenotype of two independent AP-3 subunit mutants. Fine-mapping led us to define two non-overlapping, relatively small critical regions within fly chromosome 3. The first critical region included the Atg2 gene, which encodes a conserved protein involved in autophagy. Loss of one functional copy of Atg2 ameliorated the pigmentation defects of mutants in AP-3 subunits as well as in two other genes previously implicated in LRO biogenesis, namely Blos1 and lightoid, and even increased the eye pigment content of wild-type flies. The second critical region included the ArfGAP1 gene, which encodes a conserved GTPase-activating protein with specificity towards GTPases of the Arf family. Loss of a single functional copy of the ArfGAP1 gene ameliorated the pigmentation phenotype of AP-3 mutants but did not to modify the eye pigmentation of wild-type flies or mutants in Blos1 or lightoid. Strikingly, loss of the second functional copy of the gene did not modify the phenotype of AP-3 mutants any further but elicited early lethality in males and abnormal eye morphology when combined with mutations in Blos1 and lightoid, respectively. These results provide genetic evidence for new functional links connecting the machinery for biogenesis of LROs with molecules implicated in autophagy and small GTPase regulation.     

Head length determination in bacteriophage T4: The role of the core protein P22

We have found that two different temperature-sensitive mutations in gene 22, tsA74 and ts22-2, produce high frequencies (up to 85%) of petite phage particles when grown at a permissive or intermediate temperature. Moreover, the ratio of petite to normal particles in a lysate depends upon the temperature at which the phage are grown. These petite phage particles appear to have approximately isometric heads when viewed in the electron microscope, and can be distinguished from normal particles by their sedimentation coefficient and by their buoyant density in CsCl. They are biologically active as detected by their ability to complement a co-infecting amber helper phage. Lysates of both mutants grown at a permissive temperature reveal not only a significant number of petite phage particles in the electron microscope, but also sizeable classes of wider-than-normal particles, particles having abnormally attached tails, and others having more than one tail.Striking protein differences exist between the purified phage particles of tsA74 or ts22-2 and wild-type T4. B1¹, a 61,000 molecular weight head protein, is completely absent from the phage particles of both mutants, and the internal protein IPIII¹ is present in reduced amounts as compared to wild type. The precursor to B1¹ is present in the lysates, but these mutations appear to prevent its incorporation into heads, so it does not become cleaved.The product of gene 22 (P22) is known to be the major protein of the morphogenetic core of the T4 head. Besides the mutations reported here, several mutations which affect head length have been found in gene 23, which codes for the major capsid protein (Doermann et al., 1973b). We suggest a model in which head length is determined by an interaction between the core (P22 and IPIII) and the outer shell (P23).     

Mutants of Escherichia coli Which Block Head Formation of Lambda

Five mutants of Escherichia coli K12 (lam 24, lam 25, lam 26, lam 27 and lam 646) that block head formation of λ are described. In vitro complementation tests and electron microscopy demonstrated that in these bacteria phage tails were produced normally, whereas head formation was abnormal, aberrant head-related structures being produced. In lysates prepared from lam 24, lam 25 and lam 26, monsters and empty heads without tail were the predominant structures, whereas in lysates from lam 27 and lam 646, petit λ and empty heads were the most common structures. The five lam mutations were located in two regions on the bacterial chromosome; lam 24, lam 25 and lam 26 were near the dnaB gene and lam 27 and lam 646 near the lac gene. It was suggested that the former three mutants are new isolates that belong to GroE mutants, whereas the latter two comprise a new group of mutants. Analyses of phage mutants (ov mutants) that overcome the interference by the lam 646 mutation revealed that this mutation blocks normal expression of the gene E of λ.     

Mechanism of head assembly and DNA encapsulation in Salmonella phage p22. I. Genes, proteins, structures and DNA maturation

The functions of ten known late genes are required for the intracellular assembly of infectious particles of the temperate Salmonella phage P22. The defective phenotypes of mutants in these genes have been characterized with respect to DNA metabolism and the appearance of phage-related structures in lysates of infected cells. In addition, proteins specified by eight of the ten late genes were identified by sodium dodecyl sulfate/polyacrylamide gel electrophoresis; all but two are found in the mature phage particle. We do not find cleavage of these proteins during morphogenesis.The mutants fall into two classes with respect to DNA maturation; cells infected with mutants of genes 5, 8, 1, 2 and 3 accumulate DNA as a rapidly sedimenting complex containing strands longer than mature phage length. 5^? and 8^? lysates contain few phage-related structures. Gene 5 specifies the major head structural protein; gene 8 specifies the major protein found in infected lysates but not in mature particles. 1^?, 2^? and 3^? lysates accumulate a single distinctive class of particle (“proheads”), which are spherical and not full of DNA, but which contain some internal material. Gene 1 protein is in the mature particle, gene 2 protein is not.Cells infected with mutants of the remaining five genes (10, 26, 16, 20 and 9) accumulate mature length DNA. 10^? and 26^? lysates accumulate empty phage heads, but examination of freshly lysed cells shows that many were initially full heads. These heads can be converted to viable phage by in vitro complementation in concentrated extracts. 16^? and 20^? lysates accumulate phage particles that appear normal but are non-infectious, and which cannot be rescued in vitro.From the mutant phenotypes we conclude that an intact prohead structure is required to mature the virus DNA (i.e. to cut the overlength DNA concatemer to the mature length). Apparently this cutting occurs as part of the encapsulation event.     

The Cytosolic DnaJ-like Protein Djp1p Is Involved Specifically in Peroxisomal Protein Import

The Saccharomyces cerevisiae DJP1 gene encodes a cytosolic protein homologous to Escherichia coli DnaJ. DnaJ homologues act in conjunction with molecular chaperones of the Hsp70 protein family in a variety of cellular processes. Cells with a DJP1 gene deletion are viable and exhibit a novel phenotype among cytosolic J-protein mutants in that they have a specific impairment of only one organelle, the peroxisome. The phenotype was also unique among peroxisome assembly mutants: peroxisomal matrix proteins were mislocalized to the cytoplasm to a varying extent, and peroxisomal structures failed to grow to full size and exhibited a broad range of buoyant densities. Import of marker proteins for the endoplasmic reticulum, nucleus, and mitochondria was normal. Furthermore, the metabolic adaptation to a change in carbon source, a complex multistep process, was unaffected in a DJP1 gene deletion mutant. We conclude that Djp1p is specifically required for peroxisomal protein import.     

Mechanism of head assembly and DNA encapsulation in Salmonella phage P22. II. Morphogenetic pathway

We have identified and characterized structural intermediates in phage P22 assembly. Three classes of particles can be isolated from P22-infected cells: 500 S full heads or phage, 170 S empty heads, and 240 S “proheads”. One or more of these classes are missing from cells infected with mutants defective in the genes for phage head assembly. By determining the protein composition of all classes of particles from wild type and mutant-infected cells, and examining the time-course of particle assembly, we have been able to define many steps in the pathway of P22 morphogenesis.In pulse-chase experiments, the earliest structural intermediate we find is a 240 S prohead; it contains two major protein species, the products of genes 5 and 8. Gene 5 protein (p5) is the major phage coat protein. Gene 8 protein is not found in mature phage. The proheads contain, in addition, four minor protein species, PI, P16, P20 and PX. Similar prohead structures accumulate in lysates made with mutants of three genes, 1, 2 and 3, which accumulate uncut DNA. The second intermediate, which we identify indirectly, is a newly filled (with DNA) head that breaks down on isolation to 170 S empty heads. This form contains no P8, but does contain five of the six protein species of complete heads. Such structures accumulate in lysates made with mutants of two genes, 10 and 26.Experiments with a temperature-sensitive mutant in gene 3 show that proheads from such 3^? infected cells are convertible to mature phage in vivo, with concomitant loss of P8. The molecules of P8 are not cleaved during this process and the data suggest that they may be re-used to form further proheads.Detailed examination of 8^? lysates revealed aberrant aggregates of P5. Since P8 is required for phage morphogenesis, but is removed from proheads during DNA encapsulation, we have termed it a scaffolding protein, though it may have DNA encapsulation functions as well.All the experimental observations of this and the accompanying paper can be accounted for by an assembly pathway, in which the scaffolding protein P8 complexes with the major coat protein P5 to form a properly dimensioned prohead. With the function of the products of genes 1, 2 and 3, the prohead encapsulates and cuts a headful of DNA from the concatemer. Coupled with this process is the exit of the P8 molecules, which may then recycle to form further proheads. The newly filled heads are then stabilized by the action of P26 and gene 10 product to give complete phage heads.     

Deletion of the mating-type sequences in Podospora anserina abolishes mating without affecting vegetative functions and sexual differentiation

The mating-type locus of Podospora anserina controls fusion of sexual cells as well as subsequent stages of development of the fruiting bodies. The two alleles at the locus are defined by specific DNA regions comprising 3.8 kb for mat+ and 4.7 kb for mat?, which have identical flanking sequences. Here we present the characterization of several mutants that have lost mat+-specific sequences. One mutant was obtained fortuitously and the other two were constructed by gene replacement. The mutants are deficient in mating with strains of either mat genotype but are still able to differentiate sexual reproductive structures. The loss of the mating type does not lead to any discernible phenotype during vegetative growth: in particular it does not change the life span of the strain. The mutants can recover mating ability if they are transformed with DNA containing the complete mat+ or mat? information. The transformants behave in crosses as do the reference mat+ or mat? strains, thus indicating that the transgenic mat+ and mat? are fully functional even when they have integrated at ectopic sites.     

Correlation Between the Expression of an Escherichia coli Cell Surface Protein and the Ability of the Protein to Bind to Lipopolysaccharide

The ompA gene of Escherichia coli codes for a major protein of the outer membrane. When this gene was moved between various unrelated strains (E. coli K-12 and two clinical isolates of E. coli) by transduction, the gene was expressed very poorly. Recombinants carrying “foreign” genes produced no OmpA protein which could be detected on polyacrylamide gels and became resistant to bacteriophage K3, which uses this protein as receptor. The recombinants were sensitive to host-range mutants of K3, indicating a very low level of OmpA protein was produced. When an E. coli K-12 recombinant carrying an unexpressed foreign ompA allele was subjected to two cycles of selection for an OmpA⁺ phenotype, a mutant strain was obtained which was sensitive to K3 and which expressed nearly normal levels of OmpA protein in the outer membrane. This strain carried mutations in the foreign ompA gene, as indicated both by genetic mapping and the alteration of a peptide in the mutant OmpA protein. The ability of the OmpA protein to bind to lipopolysaccharide (LPS) showed similar strain specificity, and the mutant OmpA protein which was expressed in an unrelated host showed enhanced ability to bind LPS from its new host. Thus, cell surface expression of the ompA gene appears to depend upon the ability of the gene product to bind LPS, suggesting that an interaction between the protein and LPS plays an essential role in biosynthesis of this outer membrane protein.     

The Dystrophin Complex Controls BK Channel Localization and Muscle Activity in Caenorhabditis elegans

Genetic defects in the dystrophin-associated protein complex (DAPC) are responsible for a variety of pathological conditions including muscular dystrophy, cardiomyopathy, and vasospasm. Conserved DAPC components from humans to Caenorhabditis elegans suggest a similar molecular function. C. elegans DAPC mutants exhibit a unique locomotory deficit resulting from prolonged muscle excitation and contraction. Here we show that the C. elegans DAPC is essential for proper localization of SLO-1, the large conductance, voltage-, and calcium-dependent potassium (BK) channel, which conducts a major outward rectifying current in muscle under the normal physiological condition. Through analysis of mutants with the same phenotype as the DAPC mutants, we identified the novel islo-1 gene that encodes a protein with two predicted transmembrane domains. We demonstrate that ISLO-1 acts as a novel adapter molecule that links the DAPC to SLO-1 in muscle. We show that a defect in either the DAPC or ISLO-1 disrupts normal SLO-1 localization in muscle. Consistent with observations that SLO-1 requires a high calcium concentration for full activation, we find that SLO-1 is localized near L-type calcium channels in muscle, thereby providing a mechanism coupling calcium influx with the outward rectifying current. Our results indicate that the DAPC modulates muscle excitability by localizing the SLO-1 channel to calcium-rich regions of C. elegans muscle.     

Maize streak virus genes essential for systemic spread and symptom development

The entire genome of single component geminiviruses such as maize streak virus (MSV) consists of a single-stranded circular DNA of ~2.7 kb. Although this size is sufficient to encode only three average sized proteins, the virus is capable of causing severe disease of many monocots with symptoms of chlorosis and stunting. We have identified viral gene functions essential for systemic spread and symptom development during MSV infection. Deletions and gene replacement mutants were created by site-directed mutagenesis and insertion between flanking MSV or reporter gene sequences contained in Agrobacterium T-DNA derived vectors. Following Agrobacterium-mediated inoculation of maize seedlings, the mutated MSV DNAs were excised from these binary vectors by homologous recombination within the flanking sequences. Our analyses show that the capsid gene of MSV, while not required for replication, is essential for systemic spread and subsequent disease development. The `+' strand open reading frame (ORF) located immediately upstream from the capsid ORF and predicted to encode a 10.9 kd protein was also found to be dispensable for replication but essential for systemic spread. By this analysis, MSV sequences that support autonomous replication were localized to a 1.7 kb segment containing the two viral intergenic regions and two overlapping complementary `-' strand ORFs. Despite the inability of the gene replacement mutants to spread systemically, both inoculated and newly developed leaves displayed chlorotic patterns similar to the phenotype observed in certain developmental mutants of maize. The similarity of the MSV mutant phenotype to these developmental mutants is discussed.     

Tuning of the FMN binding and oxido-reduction properties by neighboring side chains in Anabaena flavodoxin

Contribution of three regions (phosphate-binding, 50’s and 90’s loops) of Anabaena apoflavodoxin to FMN binding and reduction potential was studied. Thr12 and Glu16 did not influence FMN redox properties, but Thr12 played a role in FMN binding. Replacement of Trp57 with Glu, Lys or Arg moderately shifted E_ox/sq and E_sq/hq and altered the energetic of the FMN redox states binding profile. Our data indicate that the side chain of position 57 does not modulate E_ox/sq by aromatic stacking or solvent exclusion, but rather by influencing the relative strength of the H-bond between the N(5) of the flavin and the Asn58-Ile59 bond. A correlation was observed between the isoalloxazine increase in solvent accessibility and less negative E_sq/hq. Moreover, E_sq/hq became less negative as positively charged residues were added near to the isoalloxazine. Ile59 and Ile92 were simultaneously mutated to Ala or Glu. These mutations impaired FMN binding, while shifting E_sq/hq to less negative values and E_ox/sq to more negative. These effects are discussed on the bases of the X-ray structures of some of the Fld mutants, suggesting that in Anabaena Fld the structural control of both electron transfer steps is much more subtle than in other Flds.     

Requirement of NMB0065 for connecting assembly and export of sialic acid capsular polysaccharides in Neisseria meningitidis

Capsule expression in Neisseria meningitidis is encoded by the cps locus comprised of genes required for biosynthesis and surface translocation. Located adjacent to the gene encoding the polysialyltransferase in serogroups expressing sialic acid-containing capsule, NMB0065 is likely a member of the cps locus, but it is not found in serogroups A or X that express non-sialic acid capsules. To further understand its role in CPS expression, NMB0065 mutants were created in the serogroups B, C and Y strains. The mutants were as sensitive as unencapsulated strains to killing by normal human serum, despite producing near wild-type levels of CPS. Absence of surface expression of capsule was suggested by increased surface hydrophobicity and confirmed by immunogold electron microscopy, which revealed the presence of large vacuoles containing CPS within the cell. GC–MS and NMR analyses of purified capsule from the mutant revealed no apparent changes in polymer structures and lipid anchors. Mutants of NMB0065 homologues in other sialic acid CPS expressing meningococcal serogroups had similar phenotypes. Thus, NMB0065 (CtrG) is not involved in biosynthesis or lipidation of sialic acid-containing capsule but encodes a protein required for proper coupling of the assembly complex to the membrane transport complex allowing surface expression of CPS.     

Physical and genetic organization of Petite and Grande yeast mitochondrial DNA. II. DNA-DNA hybridization studies and buoyant density determinations

Buoyant density of mitochondrial DNA from 14 cytoplasmic petite mutants issued from the same grande yeast Saccharomyces cerevisiae was determined. Mutants that have retained the mitochondrial gene conferring resistance to erythromycin displayed higher buoyant density, while mutants that have retained the mitochondrial gene conferring resistance to chloramphenicol displayed lower buoyant density. It is inferred that the segment which carries the E^R gene has a higher G + C content than the segment which carries the C^R gene. DNA-DNA filter hybridizations were carried out systematically in different reciprocal pair-wise combinations between mtDNAs purified from various mutants and from the grande. All petites were found to be deleted in 42 to 93% of the grande sequence, depending on the mutant studied. Sequence homology between petite mtDNAs was greatest in mutants retaining common genetic markers and was least when different genetic markers were retained. Practically no hybridization was found between some C^RE^O and C^OE^R mutants. Correlations established between the extent of DNA-DNA hybridization, kinetic and genetic complexity show that a selective enrichment of gene specific sequences occurs in mtDNA of petites.