Maturation of the head of bacteriophage T4. II. Head-related, aberrant tau-particles

Three somewhat different types of particle accumulate in cells infected with a phage carrying a mutation in gene 21 (in addition to the tubular variant (polyhead) of the head). The major type is the so-called τ-particle. These particles are very fragile, associated with the cell membrane, and have a sedimentation coefficient of about 420 S. They possess no DNA if isolated, and contain predominantly the precursor proteins P23, P24, P22 and the internal protein IP III, in addition to protein P20 and several proteins of unknown genetic origin.The remainder of the particles are partially or completely filled with DNA. The ratio of τ-particles to these partially or completely filled particles depends upon the particular mutant (in gene 21) phage used. In cells infected with a phage carrying the amber mutation (N90) in gene 21, about 10% of the precursor head protein P23 is cleaved to P231, and correspondingly about 10% of the particles are partially or completely filled with DNA. In cells infected with the temperature-sensitive mutant (N8) in gene 21, about 1% of the particles are fully or partially filled, and correspondingly about 1% of the P23 is cleaved to P231. In either case, the DNA-associated particles contain predominantly the cleaved proteins P231 and IP III1, and have none of the P22 and IP III found in τ-particles. This observation, and the correlation of the amount of partially or completely filled particles with the extent of the cleavage of P23 in the lysates, strongly suggest that cleavage of the head proteins is required for DNA packaging to occur.The τ-particles have properties similar to the so-called prohead I particles which we have isolated as intermediates in wild-type head assembly (preceding paper). However, temperature shift-down experiments, using several different phage carrying temperature-sensitive mutations in gene 21, indicate that the bulk of the τ-particles cannot be used for normal phage production.     

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.     

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 : 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.     

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.     

Packaging of genomes in bacteriophages: a comparison of ssRNA bacteriophages and dsDNA bacteriophages.

In complex DNA bacteriophages like lambda, T4, T7, P22, P2, the DNA is packaged into a preformed precursor particle which sometimes has a smaller size and often a shape different from that of the phage head. This packaging mechanism is different from the one suggested for the RNA phages, according to which RNA nucleates the shell formation. The different mechanisms could be understood by comparing the genomes to be packaged: single stranded fII RNA has a very compact structure with high helix content. It might easily form quasispherical structures in solution (as seen in the electron microscope by Thach & Thach (1973)) around which the capsid could assemble. Double stranded phage DNA, on the other hand, is a rigid molecule which occupies a large volume in solution and has to be concentrated 15-fold during packaging into the preformed capsid, and the change in the capsid structure observed hereby might provide the necessary DNA condensation energy.     

Model for DNA packaging into bacteriophage T4 heads.

The mechanism of DNA packaging into bacteriophage T4 heads in vivo was investigated by glucosylation of hydroxymethylcytosine residues in a conditionally glucose-deficient host. Cytoplasmic DNA associated with partially packaged ts49 heads can be fully glucosylated, whereas DNA already packaged into these heads is shown to be resistant to glucosylation. After temperature shift and completion of arrested packaging into the reversible temperature-sensitive ts49 head, the structure of the DNA in the mature ts49 phage was investigated by restriction enzyme digestion, autoradiography, and other techniques. Such mature DNA appears to be fully glucosylated along part of its length and nonglucosylated on the remainder. Its structure suggests that the DNA is run into the head linearly and unidirectionally from one mature end and that there is little sequence specificity in that portion of the T4 DNA which first enters the capsid. This technique should be useful in investigation of the three-dimensional structure of first- and last-packaged DNA within the head; preliminary studies including autoradiography of osmotically shocked phage suggest that the DNA which first enters the head is deposited toward the center of the capsid and that the end of the DNA which first enters the head exits first upon injection. In conjunction with studies of the structure of condensed DNA, the positions and functions of T4 capsid proteins in DNA packaging, and the order of T4 packaging functions [Earnshaw and Harrison, Nature (London) 268:598-602, 1977; Hsiao and Black, Proc. Natl. Acad. Sci. U.S.A. 74:3652-3656, 1977; Müller-Salamin et al., J. Virol. 24:121-134, 1977; Richards et al., J. Mol. Biol. 78:255-259, 1973], the features described above suggest the following model: the first DNA end is fixed to the proximal apex of the head at p20 and the DNA is then pumped into the head enzymatically by proteins (p20 + p17) which induce torsion in the DNA molecule.     

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.     

Function of gene 49 of bacteriophage T4. II. Analysis of intracellular development and the structure of very fast-sedimenting DNA.

With the exception of mutants in gene 49, all mutants in phage T4 defective in the process of head filling accumulate a normal replicative DNA intermediate of 200S. Mutants in gene 49 produce a very fast-sedimenting (VFS) DNA with s values of greater than 1,000S. The intracellular development of the VFS-DNA generated in gene 49-defective phage-infected cells was followed by sedimentation analysis of crude lysates on neutral sucrose gradients. It was observed that the production of a 200S replicative intermediate is one step in the development of VFS-DNA. After restoring permissive conditions the development of the VFS-DNA can be reversed, but the 200S form is not regenerated under these conditions. The process of head filling can take place from the VFS-DNA under permissive conditions. From the absence of other components in the VFS-DNA complexes, its high resistance to shearing, its resistance against the attack of the single-strand-specific nuclease S1, and from its appearance in the electron microscope, a complex structure of tightly packed DNA is inferred. The demonstration by the electron microscope of branched DNA structures sometimes closely related to partially filled heads is taken in support of the idea that the process of head filling in gene 49-defective phage-infected cells is blocked by some steric hindrance in the DNA. In light of these results, the role of gene 49 is discussed as a control function for the clearance of these structures. A fixation procedure for cross-linking of gene 49-defective heads to the VFS-DNA allowed us to study progressive stages in the process of head filling. Electron microscopic evidence is presented which suggests that during the initial events the DNA accumulates in the vertexes of the head.     

DNA analysis of temperate bacteriophage Aa/23 isolated from Actinobacillus actinomycetemcomitans

The DNA of the temperate bacteriophage Aaφ23 isolated from the oral bacterium Actinobacillus actinomycetemcomitans was examined structurally both in the phage head and in the prophage. The DNA in phage particles comprises 44?kb linear molecules with a terminal redundancy of 1.6?kb, which represent circular permutations. Thus, DNA is packaged into phage heads by the headful mechanism. The Aaφ23 prophage is integrated into the host chromosome.     

Primary structure of internal peptide VII of T-even bacteriophages

As first shown by Hershey (1955,1957), osmotic rupture of the head of bacteriophage T2 releases, together with the DNA, an acid-insoluble protein fraction and an acid-soluble peptide fraction. Subsequent chromatographic analysis of the acid-soludle fraction of bacteriophage T4-infected Escherichia coli resolved two peptides, designated II and VII, corresponding to the peptide fraction of T2 described by Hershey (Eddleman &; Champe, 1966). Both peptides were found to be composed largely of acidic residues with only a limited number of other amino acids. The amino acid compositions predicted minimum molecular weights of 3900 (33 residues) and 2700 (23 residues) for peptides II and VII, respectively, consistent with their retardation on Sephadex G25 (Champe &; Eddleman, 1967). The formation of these peptides in T4-infected cells parallels the synthesis of late proteins. Pulse-labelling experiments showed, however, that the internal peptides are derived from acid-insoluble precursors, suggesting the probable involvement of proteolytic cleavage in their formation. The role of these peptides, if any, in phage replication is unclear. Eddleman &; Champe (1966) suggested that they may be inactive remnants of a core protein whose elimination is required for assembly. Alternatively, Laemmli &; Favre (1973) have proposed that the internal peptides may function to facilitate packaging of the DNA in the phage head.     

The transformation of τ-particles into T4 heads. I. Evidence for the conservative mode of this transformation.

Asynchroneous T4 phage head maturation includes the step of P23 cleavage: P23 of head-related τ-particles is cleaved into P23

1 This paper is part of the thesis of R. K. L. Bijlenga. It is number X of the series: “Studies on the morphopoieses of the head of phage T-even.”

of capsids with a conservative mode of transformation as evidenced by “heavy” labeling in temperature shift-down experiments with mutant 24 (tsL90). Assuming a subunit pool, data indicate in situ cleavage on individual precursor particles. The interpretation becomes less interesting when assuming a compartmentation of the membrane surface; this hypothesis is not ruled out.     

Structural aberrations in T-even bacteriophage. VI. Molecular weight of DNA from giant heads

Cummings et al. (1973) reported that whenl-canavanine was chased from a T-even. bacteriophage-infected culture with its analog,l-arginine, a new type of aberrant particle was formed. These particles, which were termed “lollipops”, had giant heads as long as 44 normal head lengths, and were filled with DNA. We have now separated these particles into different size classes ranging from about three to 13 normal head lengths and measured the molecular weight of their DNA. The DNA released from intact phage particles by neutral or alkaline detergent lysis was characterized using a recently described biophysical technique which determines DNA molecular weight from solution viscoelasticity. The maximum DNA size correlated roughly with phage head length, indicating that these giant heads were often filled with single, long DNA molecules rather than with several normal-sized molecules. Many of the heads, however, must have contained several molecules, since a large amount of DNA of less than maximum size was present. In alkali the native molecules separated into single strands of approximately the same length as that of the native molecules.     

Processing of bacteriophage lambda DNA during its assembly into heads

DNA purified from bacteriophage λ added to a cell-free extract derived from induced λ lysogens can be packaged into infectious phage particles (Kaiser & Masuda, 1973). In this paper the structure of the DNA which is the substrate for in vitro packaging and head assembly is described. The active precursor is a multichromosomal polymer that contains covalently closed cohesive end sites. Neither circular or linear DNA monomers nor polymers with unsealed cohesive ends are packaged efficiently into heads. The unit length monomer is packaged when it is either contained in the interior of a polymer (both of its ends are in cos sites) or when it has a free left end and a cos site on its right. The monomer unit with a free right end is not a substrate for packaging.A procedure is given for the purification of λ DNA fragments that contain either the left or the right cohesive end. The fragments are produced by digesting λ DNA with the site-specific Escherichia coli R₁ endonuclease; the left and right ends are separated by sedimentation through a sucrose gradient. These fragments are used to construct small polymers that have a unit length λ monomer with (1) a free left end and a closed right end, (2) a free right end and a closed left end, or (3) both ends closed in cos sites.     

Persistence and Growth of Fecal Bacteroidales Assessed by Bromodeoxyuridine Immunocapture

Extraintestinal growth of fecal bacteria can impair accurate assessment of watershed health. Anaerobic fecal bacteria belonging to the order Bacteroidales are attractive candidates for fecal source tracking because they have host-specific distributions and do not grow well in the presence of high oxygen concentrations. Growth of general and human-specific fecal Bacteroidales marker organisms in environmental samples (sewage) and persistence of the corresponding genetic markers were investigated using bromodeoxyuridine (BrdU) DNA labeling and immunocapture, followed by PCR detection. Background amplification of unlabeled controls occasionally occurred when a high number of PCR cycles was used. By using fluorescent detection of PCR products obtained after 15 cycles, which was determined to be quantitative, we enriched for BrdU-labeled DNA and did not detect unlabeled DNA. By using pure cultures of Bacteroides vulgatus, the ability of Bacteroidales bacteria to take up and incorporate BrdU into nascent DNA was confirmed. Fecal Bacteroidales organisms took up and incorporated BrdU into DNA during growth. In sewage incubated aerobically at the in situ temperature, Bacteroidales genetic marker sequences persisted for at least 24 h and Bacteroidales fecal bacteria grew for up to 24 h as well. Detection by PCR using a low, quantitative cycle number decreased the sensitivity of the assay such that we were unable to detect fecal Bacteroidales human-specific marker sequences in unlabeled or BrdU-labeled fractions, even when fluorescent detection was used. Using 30 PCR cycles with unlabeled fractions, human-specific Bacteroidales sequences were detected, and they persisted for up to 24 h in sewage. These data support the utility of BrdU labeling and immunocapture followed by length heterogeneity PCR or fluorescent detection using low numbers of PCR cycles. However, this method may not be sensitive enough to identify cells that are present at low densities in aquatic environments.     

Assembly of Bacillus subtilis phage phi29. 1. Mutants in the cistrons coding for the structural proteins.

The effect of mutations in the cistrons coding for the phage structural proteins has been studied by analyzing the phage-related structures accumulated after restrictive infection. Infection with susmutants in cistron 8, lacking both the major head and the fiber protein, does not produce any phage-related structure, suggesting a single route for the assembly of phage phi29; infection with ts mutants in this cistron produces isometric particles. Mutants is cistron 9, coding for the tail protein, TP1, produce DNA-free prolate heads with an internal core; these particles are abortive and contain the head proteins HPO, HP1 and HP3, the upper collar protein NP2 and the nonstructural proteins p7, p15 and p16. Mutants in cistron 10, coding for the upper collar protein, NP2, produce DNA-free isometric heads also with an internal core; they contain the head proteins and the nonstructural protein p7, suggesting that this protein forms the internal core. Mutants in cistrons 11 and 12, coding for the lower collar protein, NP3, and the neck appendages, NP1, respectively, give rise to the formation of DNA-containing normal capsids and DNA-free prolate particles, more rounded at the corners than the normal capsids and with an internal core; the DNA-containing 11-particles are formed by the head proteins and the upper collar protein; the DNA-free 11-particles contain, besides these proteins, the nonstructural protein p7 and a small amount of proteins p15 and 16. The DNA-containing 12-particles have all the normal phage structural proteins except the neck appendages, formed by protein NP1; the DNA-free particles are similar to the DNA-free 11-particles. After restricitive infection mutant sus14(1241) has a delayed lysis phenotype and produces a phage burst higher than normal, after artificial lysis. It produces DNA-containing particles, identical to wild-type phage, which have all the normal phage structural proteins, and DNA-free prolate particles, more rounded at the corners than the final phage particles and with an internal core; the last particles contain the same proteins as the DNA-free 11 or 12-particles. These particles could represent a prohead state, ready for DNA encapsulation. None of the DNA-containing particles have the nonstructural proteins p7, p15 or p16, suggesting that these proteins are released from the proheads upon DNA encapsulation.     

Interactions between a satellite bacteriophage and its helper

P4 is a satellite phage which relies on a helper such as P2 to supply the gene products necessary for particle construction and cell lysis (Six, 1975). P4 can activate the expression of late genes from a P2 helper phage, using a mechanism different from that employed by the helper. In the presence of P4, replication of P2 DNA is not required for late gene expression (Six & Lindqvist, 1971), and the polar effects of P2 amber mutations are suppressed.Despite its small size P4 codes for two late proteins as well as two early proteins. One of the P4 early proteins is that the product of gene α. The expression of P4 late genes is stimulated by the helper phage. Thus the P2 and P4 chromosomes exhibit reciprocal transactivation.The presence of the P4 genome causes the P2 head proteins to form a head smaller than that found after infection by P2 (Gibbs et al., 1973). P4 late proteins associate with head-like structures and may determine the small size of P4 heads.     

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.     

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.     

Survey of bromodeoxyuridine uptake among environmental bacteria and variation in uptake rates in a taxonomically diverse set of bacterial isolates

Incorporation of 5-Bromo-2′-Deoxyuridine (BrdU) into DNA can be used to target replicating bacteria in the environment, but differential uptake capacity is a potential bias. Among 23 bacterial isolates commonly found in soils, most took up BrdU, but at up to 10-fold different cell-specific rates. Combined with results from an in silico analysis of 1000 BrdU-labeled 16S rRNA gene sequences, our results demonstrate a BrdU uptake bias with no apparent relationship between taxa affiliation and ability to incorporate BrdU.