Bacteriophage T4 head morphogenesis. VII. Terminal stages of head maturation.

Several aspects of the terminal stages of T4 head maturation were investigated using ts and am mutants blocked at single steps of the assembly pathway. We had previously found that cells infected with mutants of gene 13, e.g., tsN38 and amE609, accumulated both stable (10 to 20%)- and fragile (80%)-filled head precursors (Hamilton and Luftig, 1972). Here we showed the following for such gene 13-defective, mutant-infected cells. (i) Using thin-section analysis the pool of phage precursor structures observed under nonpermissive conditions was one-third of that observed when the cells were cultured under permissive conditions. (ii) In order for complete conversion of the precursors into viable phage to occur, there were apparent requirements of metabolic energy, protein, and DNA synthesis. (iii) The intracellular DNA pool under nonpermissive conditions exhibited a 50% distribution between 63S (mature size) and 200 S (concatenate size) DNA, with the latter DNA serving as a precursor pool. Further, this DNA pool when spread onto a protein monolayer exhibited a dispersed array of DNA, strands around a core, which was less dense than that found for the greater than 1,000S DNA concatenate isolated from gene 49-defective infected cells. (iv) When precuations were taken to stabilize the head precursors, such as lysis of the cells into glutaraldehyde, there was a 30% increase in the yield of 1,200S filled heads. Correlating these results and previous results concerning gene 49-defective unfilled heads, we propose that there are several forms of gene 13 fragile head precursors which serve as intermediates between gene 49 unfilled heads and gene 13 stable filled heads. We cannot, however, rule out the possibility that all gene 13-defective heads represent a single class of unstable particles, which decay slowly. In either case, we have shown that gene 13-defective particles are unstable to some degree inside the cell and are highly unstable outside the cell; yet all particles can still be efficiently converted to phage in vivo.     

Bacteriophage T4 Head Morphogenesis II. Studies on the Maturation of Gene 49-Defective Head Intermediates

An investigation into the metabolic requirements for maturation of gene 49-defective heads indicated that adenosine triphosphate energy and continued deoxyribonucleic acid (DNA) but not ribonucleic acid synthesis were needed. The fate of DNA present at restrictive temperatures (41.5 C) in tsC9 (gene 49)-infected cells was also examined. After lysis of infected cells, the 12 to 32% deoxyribonuclease-resistant DNA associated with isolated gene 49-defective heads was found to be attached to a deoxyribonuclease-sensitive complex associated with the debris. Pulsechase experiments where (3)H-thymidine was used to label the DNA at 41.5 C suggested that more DNA from this pool was present in phage recovered after rescue of the gene 49 function than could be accounted for by the deoxyribonuclease-resistant portion. Further, when these experiments were repeated with an additional density shift ((15)N(13)C-glucose to (14)N(12)C-glucose), the DNA extracted from phage rescued at 10 min after the temperature shift-down was found to be 90% conserved. These results suggest a model whereby DNA packaging into capsid precursors is separated from DNA replication and the energy from DNA synthesis provides the driving force for packaging. Pulse-chase, temperature-shift experiments with E920g (gene 66) or E920g;tsC9 mutant-infected cells showed that gene (49, 66)-defective heads, which were isolated as small, isometric-shaped unfilled heads, were a precursor to "petite" phage. This suggests that the maturation process is independent of the size and shape of the head membrane. Similar experiments with the double mutant tsC9;amN120 indicate that gene 49-defective heads can also be filled in the absence of tails.     

Bacteriophage T4 head morphogenesis : On the nature of gene 49-defective heads and their role as intermediates

Following infection under non-permissive conditions, T4 mutants defective in gene 49 accumulate structures which appear in the electron microscope to be empty phage heads. These structures are seen in extracts prepared under a variety of conditions, as well as in sections of the mutant-infected cells. The 49-defective heads (300 s) can be separated from phage particles (1000 s) by sedimentation through a sucrose gradient. A temperature-sensitive gene 49 mutant, tsC9, accumulates 300 s heads following infection at 41.5 °C, but can be “rescued” by a shift-down to 25 °C during the latter half of the latent period. Evidence from pulse-chase isotopic labeling experiments suggests that the 49-defective heads are intermediates in head formation. ¹⁴C-Labeled lysine, incorporated into the 300 s fraction at 41.5 °C, is rapidly and almost quantitatively transferred into the 1000 s phage particle fraction following a chase with an excess of unlabeled lysine and a shift to low temperature. The same result is observed when puromycin (200 μg/ml.) or chloramphenicol (200 μg/ml.) is added to the culture before temperature shift, suggesting that the inactive gene 49 product produced at high temperature becomes active at low temperature. In pulse-chase experiments carried out with wild-type T4-infected cells during the latter half of the latent period, the labeling kinetics of the 300 s and phage particle fractions support a precursor-product relationship. Conservation of the 300 s head structures during conversion to phage is demonstrated by ¹³C-¹⁵N density labeling of tsC9-infected cells at 41.5 °C followed by transfer to ¹²C-¹⁴N medium, shift to low temperature, isolation and lysis of the phage particles formed and centrifugation of the phage ghosts to equilibrium in CsCl solution.     

Maturation of the head of bacteriophage T4. III. DNA packaging into preformed heads

An estimate was made of the amount of DNA packaged into gene 49-defective heads when P49 is activated by a temperature shift. The uptake of DNA into preformed heads following activation of P49 was studied using bromo-deoxyuridine as a label. The rate of inactivation by visible light of the phage matured in the presence of BrdU as well as their buoyant density in CsCl, indicate that over half of the particles package, on the average, at least 25% of the DNA complement following P49 activation. This is a minimum estimate, since the BrdU-labeled DNA has to compete with unlabeled DNA. Analysis on alkaline sucrose gradients of the size of the DNA extracted from phage matured in the presence of BrdU following irradiation reveals that extended irradiation at 313 nm breaks the DNA close to half of its original size. These experiments clearly show that up to half of the DNA can be packaged into the preformed heads made at high temperature following activation of the product of gene 49 (P49), strongly supporting the pathway for phage head maturation described by Laemmli &; Favre (1973).The so-called τ-particles, which accumulate in 24-defective cells, can serve as precursors of the mature phage (Bijlenga et al., 1973). We have measured the uptake of BrdU-labeled DNA into τ-particles during their maturation. We find that a very large proportion of DNA made after activation of P24 is apparently incorporated into preformed τ-particles as these particles are converted into mature heads. This indicates that the τ-particles contain very little or no DNA prior to P24 activation and supports the pathway described by Laemmli &; Favre (1973).     

Bacteriophage T4 Head Morphogenesis IV. Comparison of Gene 16-, 17-, and 49-Defective Head Structures2

Defective heads present in extracts of bacteriophage T4 gene 16, 17, or 49 mutant-infected cells have been characterized. All appeared as empty shells when examined by negative-stain electron microscopy and showed essentially the same polypeptide pattern on sodium dodecyl sulfate-acrylamide gels. However, when analyzed by several other methods, gene 16- and 17-defective heads were shown to differ markedly from phage heads present in gene 49-defective extracts. First, the gene 16- and 17-defective structures were found to possess a large number of attached tails (50%, rather than about 5%). Second, they contained less nuclease-resistant deoxyribonucleic acid (DNA) (3 versus 18% of a phage equivalent), had a smaller sedimentation coefficient (240 versus 315S), and a lighter density (1.31 vs. 1.34 g/ml) than gene 49-defective heads. Third, they were not attached to the intracellular DNA pool through a deoxyribonuclease-sensitive linkage. Finally, 8-nm diameter capsomers were clearly revealed on the surface of many gene 16- and 17-defective structures. There was a total of 305 ± 25 capsomers per particle, which yielded an approximate molecular weight of 84 × 10⁶ for these heads. The capsomers were presumably not seen on gene 49-defective heads because of the large amount (18%) of associated DNA.     

Maturation of the head of bacteriophage T4. I. DNA packaging events

Pulse-chase experiments in wild-type and mutant phage-infected cells provide evidence that the following particles called prohead I, II and III are successive precursors to the mature heads. The prohead I particles contain predominantly the precursor protein P23 and possibly P22 (mol. wt 31,000) and IP III (mol. wt 24,000) and have an s value of about 400 S. Concomitantly with the cleavage of most of P23 (mol. wt 55,000) to P231 (mol. wt 45,000), they are rapidly converted into prohead II particles which sediment with about 350 S. The prohead II particles contain, in addition to P231, the major constituents of the viral shella—a core consisting of proteins P22 and IP III. In cell lysates, prohead I and prohead II particles contain no DNA in a DNase-resistant form and are not bound to the replicative DNA. We cannot, however, positively rule out the possibility that these particles may have contained some DNA while in the cells.The prohead II particles are in turn converted into particles which sediment with about 550 S after DNase treatment (prohead III). During this conversion about 50% of normal DNA complement becomes packaged in a DNase-resistant form, and roughly 50% of the core proteins P22 and IP III are cleaved. In lysates the prohead III particles are attached to the replicative DNA. The prohead III particle appears to be the immediate precursor of the full mature head (1100 S). Cleavage of protein P22 to small polypeptides and conversion of IP III IP III1 are completed at this time. No precursor proteins are found in the full heads. Studies with various mutant phage showed that the prohead II to III conversion is blocked by mutations in genes 16 and 17 and that the conversion of the prohead III particles to the mature heads is blocked by mutations in gene 49. Cleavage of the head proteins, however, occurs normally in these mutant-infected cells. We conclude that the cleavage of the major component of the viral shell, P23, into P231 precedes the DNA packaging event, whereas cleavage of the core proteins P22 and IP III appears to be intimately linked to the DNA packaging event. Models relating the cleavage processes to DNA encapsulation are discussed.     

Morphogenesis of bacteriophage lambda deletion mutants. I. Abnormal head-related structures produced in normal Escherichia coli.

Late in the morphogenesis of bacteriophage lambda, DNA condenses into the nascent head and is cut from a concatemeric replicative intermediate by a nucleolytic function, Ter, acting at specific sites, called cos. As a result of this process, heads of lambda deletion mutants contain less DNA than those of the wild-type phage. It has been reported that phage with very large deletions (22% of the genome or more) grow poorly but that normal growth can be restored by the non-specific addition of DNA to the genome. This finding implies that DNA content may exert a physical effect on some stage of head assembly.We have investigated the effects of two long deletions, b221 and tdel33, on head assembly. Bacteria infected with the mutants were lysed with non-ionic detergent under conditions favoring stabilization of labile structures containing condensed DNA. It has proved possible to isolate two aberrant head-related structures produced by the deletion mutants. One of these (“overfilled heads”) contains DNA which is longer than the deletion mutant genome and is about the same size as that found in wild-type heads. These structures appear to be unable to attach tails. The second type of structure (“incompletely filled heads”) contains a short piece of DNA, 40% of the length of the mutant genome. The incompletely filled heads are found both with and without attached tails. Both of these abnormal structures are initially attached to the replicating DNA but are released by treatment with DNAase. The nature of these abnormal structures indicates that very small genomes affect a late stage of head morphogenesis, after the DNA is complexed with a capsid of normal size. The results presented suggest that underfilling of the capsid interferes with the ability of the Ter function to properly cleave cos.     

Involvement of Gene 49 in Recombination of Bacteriophage T4

The role of T4 gene 49 in recombination was investigated using its conditional-lethal amber (am) and temperature-sensitive (ts) mutants. When measured in genetic tests, defects in gene 49 produced a recombination-deficient phenotype. However, DNA synthesized in cells infected with a ts mutant (tsC9) at a nonpermissive temperature appeared to be in a recombinogenic state: after restitution of gene function by shifting to a permissive temperature, the recombinant frequency among progeny increased rapidly even when DNA replication was blocked by an inhibitor. Growth of a gene 49-defective mutant was suppressed by an additional mutation in gene uvsX, but recombination between rII markers was not.     

Bacteriophage T4 Head Morphogenesis III. Some Novel Properties of Gene 13-Defective Heads

The nature of phage precursors in gene 13-defective infected cells was studied by electron microscopy and pulse-chase isotopic labeling experiments. Our results suggest that both stable (20%) and fragile (70%) filled-head precursors accumulated in the absence of gene 13 product. Upon extraction, the fragile heads were found to lose most of their deoxyribonucleic acid and appeared unfilled with an average density of 1.34 g/cm(3) and a sedimentation coefficient of 300S. These unfilled heads differed from empty gene 13-defective heads which did not have any associated deoxyribonucleic acid and banded at an average density of 1.31 g/cm(3). Furthermore, it was found that a tsN38 (temperature-sensitive mutant in gene 13)- infected culture maintained at 41.5 C for increasing times led to a decrease in specific infectivity of 1,000S phagelike particles. Electron microscopy of these particles revealed that the decreased infectivity was due to an improper union of head and tails.     

Nanovariant RNAs: nucleotide sequence and interaction with bacteriophage Qbeta replicase

Gene 2 amber mutants of bacteriophage T4 grown on su^? hosts produce whole particles of which less than 0.5% are infective on su⁺ hosts. Although the DNA of such particles is full-sized and un-nicked, it is degraded to acid-soluble fragments after infection of exo V⁺ hosts. This breakdown does not occur on exo V^? deficient hosts, and such hosts are fully permissive for gene 2-defective particles. We have now determined that giant-headed, gene 2-defective particles containing several genome lengths of DNA per head are fully infective on exo V⁺ hosts even though part of the parental DNA is degraded to acid-soluble fragments early after infection. Restriction of gene 2-defective particles must therefore be due to exonucleolytic degradation of the incoming DNA. If the parental DNA is of sufficient length to enable a complete genome to survive this degradation before production of anti-exoV, such particles are now infective.     

Bacteriophage G4 DNA synthesis in temperature-sensitive dna mutants of Escherichia coli.

The synthesis of bacteriophage G4 DNA was examined in temperature-sensitive dna mutants under permissive and nonpermissive conditions. The infecting single-stranded G4 DNA was converted to the parental replicative form (RF) at the nonpermissive temperature in infected cells containing a temperature sensitive mutation in the dnaA, dnaB, dnaC, dnaE, or dnaG gene. The presence of 30 mug of chloramphenicol or 200 mug of rifampin per ml had no effect on parental RF synthesis in these mutants. Replication of G4 double-stranded RF DNA occurred at a normal rate in dnaAts cells at the nonpermissive temperature, but the rate was greatly reduced in cells containing a temperature-sensitive mutation in the dnaB, dnaC, dnaE, or dnaG gene. RF DNA replicated at normal rates in revertants of these dna temperature-sensitive host cells. The simplest interpretation of these observations is that none of the dna gene products tested is essential for the synthesis of the complementary DNA strand on the infecting single-stranded G4 DNA, whereas the dnaB, dnaC, dnaE, (DNA polymerase III), and dnaG gene products are all essential for replication of the double-stranded G4 RF DNA. The alternate possibility that one or more of the gene products are actually essential for G4 parental RF synthesis, even though this synthesis is not defective in the mutant hosts, is also discussed.     

Replication of herpesvirus DNA. V. Maturation of concatemeric DNA of pseudorabies virus to genome length is related to capsid formation.

The maturation of pseudorabies virus DNA from the replicative concatemeric form to molecules of genome length was examined using nine DNA+ temperature-sensitive mutants of pseudorabies virus, each belonging to a different complementation group. At the nonpermissive temperature, cells infected with each of the mutants synthesized concatemeric DNA. Cleavage of the concatemeric DNA to genome-length viral DNA was defective in all the DNA+ ts mutants tested, indicating that several viral gene products are involved in the DNA maturation process. In none of the ts mutant-infected cells were capsids with electron-dense cores (containing DNA) formed. Empty capsids with electron-translucent cores were, however, formed in cells infected with six of the nine temperature-sensitive mutants; in cells infected with three of the mutants, no capsid assembly occurred. Because these three mutants are deficient both in maturation of DNA and in the assembly of viral capsids, we conclude that maturation of viral DNA is dependent upon the assembly of capsids. In cells infected with two of the mutants (tsN and tsIE13), normal maturation of viral DNA occurred after shiftdown of the cells to the permissive temperature in the presence of cycloheximide, indicating that the temperature-sensitive proteins involved in DNA maturation became functional after shiftdown. Furthermore, because cycloheximide reduces maturation of DNA in wild-type-infected cells but not in cells infected with these two mutants, we conclude that a protein(s) necessary for the maturation of concatemeric DNA, which is present in limiting amounts during the normal course of infection, accumulated in the mutant-infected cells at the nonpermissive temperature. Concomitant with cleavage of concatemeric DNA, full capsids with electron-dense cores appeared after shiftdown of tsN-infected cells to the permissive temperature, indicating that there may be a correlation between maturation of DNA and formation of full capsids. The number of empty and full capsids (containing electron-dense cores) present in tsN-infected cells incubated at the nonpermissive temperature, as well as after shiftdown to the permissive temperature in the presence of cycloheximide, was determined by electron microscopy and by sedimentation analysis in sucrose gradients. After shiftdown to the permissive temperature in the presence of cycloheximide, the number of empty capsids present in tsN-infected cells decreased with a concomitant accumulation of full capsids, indicating that empty capsids are precursors to full capsids.     

Genetic control of formation of very fast sedimenting DNA of bacteriophage T4

Summary Formation of very fast sedimenting DNA (VFS-DNA) in cells of Escherichia coli infected with phage T4 carrying a defect in gene 49 was differentially affected by a secondary mutation in gene 30 or 46; a mutation of gene 46 markedly reduced formation of VFS-DNA, whereas that of gene 30 did not.     

Regulation of coliphage f1 single-stranded DNA synthesis by a DNA-binding protein

Control of single-strand DNA synthesis in coliphage f1 was studied with the use of mutants which are temperature sensitive in gene 2, a gene essential for phage DNA replication. Cells were infected at a restrictive temperature with such a mutant, and the DNA synthesized after a shift to permissive temperature was examined. When cells were held at 42 °C for ten or more minutes after infection, only single-stranded DNA was synthesized immediately after the shift to permissive temperature. This indicated that the accumulation of a pool of double-stranded, replicative form DNA molecules is not an absolute requirement for the synthesis of single-stranded DNA, although replicative form DNA accumulation precedes single-strand synthesis in cells infected with wild-type phage. Cells infected at restrictive temperature with the mutant phage do not replicate the infecting DNA, but do accumulate a substantial amount of gene 5 protein, a DNA-binding protein essential for single-strand synthesis. It is proposed that this accumulated gene 5 protein, by binding to the limited number of replicating DNA molecules formed following the shift to the permissive temperature, acts to prevent the synthesis of double-stranded replicative form DNA, thus causing the predominant appearance of single strands. This explanation implies an intermediate common to both single and double-stranded DNA synthesis. The kinetics of gene 5 protein synthesis indicates that it is the ratio of the gene 5 protein to replicating DNA molecules which determines whether an intermediate will synthesize double or single-stranded DNA.     

Rescue of abortive T7 gene 2 mutant phage infection by rifampin.

Infection of Escherichia coli with T7 gene 2 mutant phage was abortive; concatemeric phage DNA was synthesized but was not packaged into the phage head, resulting in an accumulation of DNA species shorter in size than the phage genome, concomitant with an accumulation of phage head-related structures. Appearance of concatemeric T7 DNA in gene 2 mutant phage infection during onset of T7 DNA replication indicates that the product of gene 2 was required for proper processing or packaging of concatemer DNA rather than for the synthesis of T7 progeny DNA or concatemer formation. This abortive infection by gene 2 mutant phage could be rescued by rifampin. If rifampin was added at the onset of T7 DNA replication, concatemeric DNA molecules were properly packaged into phage heads, as evidenced by the production of infectious progeny phage. Since the gene 2 product acts as a specific inhibitor of E. coli RNA polymerase by preventing the enzyme from binding T7 DNA, uninhibited E. coli RNA polymerase in gene 2 mutant phage-infected cells interacts with concatemeric T7 DNA and perturbs proper DNA processing unless another inhibitor of the enzyme (rifampin) was added. Therefore, the involvement of gene 2 protein in T7 DNA processing may be due to its single function as the specific inhibitor of the host E. coli RNA polymerase.     

Bypass of a primase requirement for bacteriophage T4 DNA replication in vivo by a recombination enzyme, endonuclease VII.

A primase, the product of phage T4 gene 61, is required to initiate synthesis of Okazaki pieces and to allow bidirectional replication from several T4 origins. However, primase-defective T4 gene 61 mutants are viable. In these mutants, leading-strand DNA synthesis starts at the same time as in wild type infections, but, in contrast to wild type, initiation is unidirectional and the first replicative intermediates are large displacement loops. Rapid double-strand DNA replication occurs later after infection, generating multiple branched concatemers, which are cut and packaged into viable progeny particles, as in wild-type T4. Evidence is presented that this late double-strand DNA replication requires functional endonuclease VII (endo VII), the product of the T4 gene 49. We propose that endo VII can provide a backup mechanism when primase is defective, because it cuts recombinational junctions, generating 3' ends. These ends can prime DNA synthesis to copy the DNA strands that had been displaced during the initial origin-dependent replication. We explain the DNA-delay phenotype and the commonly observed temperature dependence of DNA replication in primase-deficient gene 61 mutants as a consequence of temperature-dependent translational control of gene 49 expression. In the presence or absence of functional primase endo VII is essential for correct packaging of DNA. The powerful selection that keeps the function of endo VII and expression of its gene at levels that are optimal for T4 development determines both the efficiency and the limitations of the bypass mechanism.     

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).     

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.     

Isolation and characterization of temperature-sensitive mutants of the Staphylococcus aureus dnaC gene

A protein encoded by the Staphylococcus aureus dnaC gene has 44% and 58% homology with Escherichia coli DnaB and Bacillus subtilis DnaC replicative DNA helicases, respectively. We identified five mutant strains whose temperature-sensitive colony formation phenotypes were complemented by the dnaC gene. DNA replication in these mutants has a fast-stop phenotype, indicating that the S. aureus dnaC gene encodes the replicative DNA helicase required for the elongation step. These mutants were also sensitive to UV irradiation, suggesting that the dnaC gene is involved in DNA repair. The number of viable mutant cells decreased at a non-permissive temperature, suggesting that S. aureus DnaC helicase is a promising target for antibiotics providing bactericidal effects.