Defective Concatemer Formation in Cells Infected with Deoxyribonucleic Acid-Delay Mutants of Bacteriophage T4

Nonpermissive host cells infected with phage T4 mutants in genes 52, 39, and 58 through 61 are shown to form short intracellular single-stranded deoxyribonucleic acid in contrast to wild-type infected cells, which form dimers and trimers of T4 genome size.     

Partial Suppression of Bacteriophage T4 Ligase Mutations by T4 Endonuclease II Deficiency: Role of Host Ligase

Endonuclease II-deficient, ligase-deficient double mutants of phage T4 induce considerably more deoxyribonucleic acid (DNA) synthesis after infection of Escherichia coli B than does the ligase-deficient single mutant. Furthermore, the double mutant can replicate 10 to 15% as well as wild-type T4, whereas the single mutant fails to replicate. When the E. coli host is also deficient in ligase, the double mutant resembles the single mutant. The results indicate that host ligase can substitute for phage ligase when the host DNA is not attacked by the phage-induced endonuclease II.     

Host- and Phage-Mediated Repair of Radiation Damage in Bacteriophage T4

T4+ exhibits increased ultraviolet sensitivity on derivatives of Escherichia coli K12 or B lacking deoxyribonucleic acid (DNA) polymerase I. However, the sensitivity of T4v is not affected by the absence of host DNA polymerase. T4x and T4y also show increased sensitivity on DNA polymerase-deficient strains, but to a lesser extent than observed with wild-type T4. When T4x or T4y, but not T4+, are plated on a double mutant lacking both DNA polymerase and the uvrA gene product, a partial suppression of the polymerase effect is observed. Host ligase appears to be able to suppress to some extent the T4y phenotype but has no effect on wild-type T4 or other T4 mutants. T4xv incubated in E. coli B or B(s-1) in the presence of chloramphenicol (50 mug/ml) shows increased resistance over directly plated irradiated phage. Increased survival under the same conditions was not observed with T4+ or other T4 mutants. The repair of X-ray-damaged T4 was investigated by examining survival curves of T4+, T4x, T4y, T4ts43, and T4ts30. The repair processes were further defined by observing the effects of plating irradiated phage on various hosts including strains lacking DNA polymerase I or polynucleotide ligase. Two classes of effects were observed. Firstly, the x and y gene products seem to be involved in a repair system utilizing host ligase. Secondly, in the absence of host DNA polymerase, phage sensitivity is increased in an unknown manner which is enhanced by the presence of host uvrA gene product.     

A quorum-sensing signaling system essential for genetic competence in Streptococcus mutans is involved in biofilm formation

In a previous study, a quorum-sensing signaling system essential for genetic competence in Streptococcus mutans was identified, characterized, and found to function optimally in biofilms (Li et al., J. Bacteriol. 183:897-908, 2001). Here, we demonstrate that this system also plays a role in the ability of S. mutans to initiate biofilm formation. To test this hypothesis, S. mutans wild-type strain NG8 and its knockout mutants defective in comC, comD, comE, and comX, as well as a comCDE deletion mutant, were assayed for their ability to initiate biofilm formation. The spatial distribution and architecture of the biofilms were examined by scanning electron microscopy and confocal scanning laser microscopy. The results showed that inactivation of any of the individual genes under study resulted in the formation of an abnormal biofilm. The comC mutant, unable to produce or secrete a competence-stimulating peptide (CSP), formed biofilms with altered architecture, whereas the comD and comE mutants, which were defective in sensing and responding to the CSP, formed biofilms with reduced biomass. Exogenous addition of the CSP and complementation with a plasmid containing the wild-type comC gene into the cultures restored the wild-type biofilm architecture of comC mutants but showed no effect on the comD, comE, or comX mutant biofilms. The fact that biofilms formed by comC mutants differed from the comD, comE, and comX mutant biofilms suggested that multiple signal transduction pathways were affected by CSP. Addition of synthetic CSP into the culture medium or introduction of the wild-type comC gene on a shuttle vector into the comCDE deletion mutant partially restored the wild-type biofilm architecture and further supported this idea. We conclude that the quorum-sensing signaling system essential for genetic competence in S. mutans is important for the formation of biofilms by this gram-positive organism.     

Identification and preliminary characterization of a mutant defective in the bacteriophage T4-induced unfolding of the Escherichia coli nucleoid.

The nucleoids of Escherichia coli S/6/5 cells are rapidly unfolded at about 3 min after infection with wild-type T4 bacteriophage or with nuclear disruption deficient, host DNA degradation-deficient multiple mutants of phage T4. Unfolding does not occur after infection with T4 phage ghosts. Experiments using chloramphenicol to inhibit protein synthesis indicate that the T4-induced unfolding of the E. coli chromosomes is dependent on the presence of one or more protein synthesized between 2 and 3 min after infection. A mutant of phage T4 has been isolated which fails to induce this early unfolding of the host nucleoids. This mutant has been termed "unfoldase deficient" (unf-) despite the fact that the function of the gene product defective in this strain is not yet known. Mapping experiments indicate that the unf- mutation is located near gene 63 between genes 31 and 63. The folded genomes of E. coli S/6/5 cells remain essentially intact (2,000-3,000S) at 5 min after infection with unfoldase-, nuclear disruption-, and host DNA degradation-deficient T4 phage. Nuclear disruption occurs normally after infection with unfoldase- and host DNA degradation-deficient but nuclear disruption-proficient (ndd+), T4 phage. The host chromosomes remain partially folded (1,200-1,800S) at 5 min after infection with the unfoldase single mutant unf39 x 5 or an unfoldase- and host DNA degradation-deficient, but nuclear disruption-proficient, T4 strain. The presence of the unfoldase mutation causes a slight delay in host DNA degradation in the presence of nuclear disruption but has no effect on the rate of host DNA degradation in the absence of nuclear disruption. Its presence in nuclear disruption- and host DNA degradation-deficient multiple mutants does not alter the shutoff to host DNA or protein synthesis.     

Growth Inhibition and Appearance of the Membranous Structure in Escherichia coli Infected with Bacteriophage fd

Thirty-six mutants of fd, a virus that infects but does not kill Escherichia coli, were isolated; 35 mutants were categorized into six complementation groups. Abortive infection with mutants in genes 1, 3, 4, 5, and 6, but not in gene 2, produced a cessation of host cell growth, generally linked to low burst size and to the formation of aberrant intracytoplasmic membranous structures. The membranous structure was studied during infection with various phage and hosts. Appearance of the membranous structure was linked specifically to incomplete phage maturation at the cell membrane, rather than solely to the inhibition of host cell growth or to infection with mutant phage, since (i) in one host, cell growth was inhibited, but no membranous structure developed; and (ii) when antibody against virus was added to cells infected with wild-type phage, phage extrusion was inhibited, cell growth stopped, and the membranous structure once again developed.     

Specific Suppression of Mutations in Genes 46 and 47 by das, a New Class of Mutations in Bacteriophage T4D

Mutants in T4 genes 46 and 47 exhibit early cessation of deoxyribonucleic acid (DNA) synthesis ("DNA arrest") and decreased synthesis of late proteins and phage. In addition, mutants in genes 46 and 47 fail to degrade host DNA to acidsoluble products. It is shown here that this complex phenotype can be partially suppressed by mutation of a T4 gene external to genes 46 and 47 which has been named das for "DNA arrest suppressor." The das mutations were discovered as third-site mutations in spontaneous pseudorevertants of [46, 47] mutants; the pseudorevertants make small plaques on Escherichia coli B, whereas [46, 47] mutants make none. The [das, 46, 47] triple mutant exhibits increased DNA, late protein, and viable phage production compared to the double mutant [46, 47]. The [das, 46, 47] mutant also degrades more of the host DNA to acid-soluble products than does the [46, 47] mutant. The suppressor effect of the das mutation appears to be gene-specific: it suppresses both amber and temperature-sensitive mutations in genes 46 and 47 and does not suppress amber mutations in any of the other genes tested. The [das] single mutants make normal-sized plaques on E. coli B and exhibit nearly normal host DNA degradation, DNA synthesis, late protein synthesis, and viable phage production. The das mutations either define a new gene between genes 33 and 34 or are special mutations within gene 33.     

Altered biological properties of cell membranes in Escherichia coli dnaA and seqA mutants.

We present evidence that biological properties of cell membranes are altered in dnaA and seqA mutants of Escherichia coli relative to wild-type bacteria. We found that bacteriophage lambda forms extremely large plaques on the dnaA seqA double mutants. On the single mutants, dnaA and seqA, the plaques are also bigger than those formed on the wild-type host. However, no significant differences in intracellular phage lambda development were observed between wild-type and mutant hosts, indicating that differences in burst size do not account for the observed differences in plaque size. On the other hand, more efficient release of the phage lytic proteins and/or higher sensitivity of the cell membranes to these proteins may result in more efficient cell lysis. We found that the efficiency of adsorption of bacteriophage lambda to the dnaA seqA mutant cells is decreased at 0 degrees C , but not at 30 degrees C, relative to the wild-type strain. A considerable increase in the permeability of membranes of the mutant cells for beta-galactosidase is demonstrated. The dnaA and seqA mutants are more sensitive to ethanol (an organic solvent) than wild-type bacteria, and the seqA strain and the double mutant dnaA seqA are very sensitive to deoxycholate (a detergent). We conclude that lesions in the genes dnaA and seqA result in alterations in cell membranes, such that the permeability and possibly also other properties of the membranes are significantly altered relative to wild-type bacteria.     

Growth of a Dihydrofolate Reductaseless Mutant of Bacteriophage T4

A mutant of bacteriophage T4 was isolated which was unable to induce virus-specific dihydrofolate reductase in infected cells. The mutant was able to form several other early enzymes of pyrimidine metabolism. Growth of the mutant in a wild-type host, Escherichia coli B, was compared with that of the parent strain, T4BO(1), and T4td8, a mutant which lacks the ability to induce thymidylate synthetase. Growth studies were carried out in minimal medium, which gave higher growth rates and phage yields than the supplemented media used in previous studies. The reductase mutant formed deoxyribonucleic acid and plaque-forming particles at a rate slightly higher than the synthetase mutant but 1.5-to 2-fold lower than that of the wild-type phage under all conditions studied. The addition of thymine to a culture infected by the mutant increased the growth rate significantly, suggesting that the genetic lesion leads to a partial thymidylate deficiency. Like other viral genes controlling steps in thymidylate metabolism, the dihydrofolate reductase gene appears to be useful but not completely essential for growth.     

Mutants in a Nonessential Gene of Bacteriophage T4 Which Are Defective in the Degradation of Escherichia coli Deoxyribonucleic Acid

Wild-type bacteriophage T4 was enriched for mutants which fail to degrade Escherichia coli deoxyribonucleic acid (DNA) by the following method. E. coli B was labeled in DNA at high specific activity with tritiated thymidine ((3)H-dT) and infected at low multiplicity with unmutagenized T4D. At 25 min after infection, the culture was lysed and stored. Wild-type T4 degrades the host DNA and incorporates the (3)H-dT into the DNA of progeny phage; mutants which fail to degrade the host DNA make unlabeled progeny phage. Wild-type progeny are eventually inactivated by tritium decay; mutants survive. Such mutants were found at a frequency of about 1% in the survivors. Eight mutants are in a single complementation group called denA located near gene 63. Four of these mutants which were examined in detail leave the bulk of the host DNA in large fragments. All eight mutants exhibit much less than normal T4 endonuclease II activity. The mutants produce somewhat fewer phage and less DNA than does wild-type T4.     

Gyrase-dependent initiation of bacteriophage T4 DNA replication: interactions of Escherichia coli gyrase with novobiocin, coumermycin and phage DNA-delay gene products.

Wild-type bacteriophage T4 and DNA-delay am mutants defective in genes 39, 52, 60 and 58–61 were tested for intracellular sensitivity to the antibiotics coumermycin and novobiocin, drugs which inhibit the DNA gyrase of Escherichia coli. Treatment with these antibiotics drastically reduced the characteristic growth of gene 39, 52 and 60 DNA-delay am mutants in E. coli lacking an amber suppressor (su^?). Wild-type phage-infected cells were unaffected by the drugs while the burst size of a gene 58–61 mutant was affected to an intermediate extent. A su^?E. coli strain which is resistant to coumermycin due to an altered gyrase permitted growth of the DNA-delay am mutants in the presence of the drug. Thus, the characteristic growth of the DNA-delay am mutants in an su^? host apparently depends on the host gyrase. An E. coli himB mutant is defective in the coumermycin-sensitive subunit of gyrase (H. I. Miller, personal communication). Growth of the gene 39, 52 and 60 am mutants was inhibited in the himB mutant while the gene 58–61 mutant and wild-type T4 showed small reductions in burst size in this host. Experiments with nalidixic acid-sensitive and resistant strains of E. coli show that wild-type phage T4 requires a functional nalA protein for growth.Novobiocin and coumermycin inhibit phage DNA synthesis in DNA-delay mutant-infected su^?E. coli if added during the early logarithmic phase of phage DNA synthesis. The gene 58–61 mutant showed the smallest inhibition of DNA synthesis in the presence of the drugs. Addition of the drugs during the late linear phase of phage DNA synthesis had no effect on further synthesis in DNA-delay mutant-infected cells. Coumermycin and novobiocin had no effect on DNA synthesis in wild-type-infected cells regardless of the time of addition of the antibiotics. Models are considered in which the DNA-delay gene products either form an autonomous phage gyrase or interact with the host gyrase and adapt it for proper initiation of phage DNA replication.     

Isolation of Bacteriophage T4 Mutants Defective in the Ability to Degrade Host Deoxyribonucleic Acid

A method was devised for identifying nonlethal mutants of T4 bacteriophage which lack the capacity to induce degradation of the deoxyribonucleic acid (DNA) of their host, Escherichia coli. If a culture is infected in a medium containing hydroxyurea (HU), a compound that blocks de novo deoxyribonucleotide biosynthesis by interacting with ribonucleotide reductase, mutant phage that cannot establish the alternate pathway of deoxyribonucleotide production from bacterial DNA will fail to produce progeny. The progeny of 100 phages that survived heavy mutagenesis with hydroxylamine were tested for their ability to multiply in the presence of HU. Four of the cultures lacked this capacity. Cells infected with one of these mutants, designated T4nd28, accumulated double-stranded fragments of host DNA with a molecular weight of approximately 2 x 10(8) daltons. This mutant failed to induce T4 endonuclease II, an enzyme known to produce single-strand breaks in double-stranded cytosine-containing DNA. The properties of nd28 give strong support to an earlier suggestion that T4 endonuclease II participates in host DNA degradation. The nd28 mutation mapped between T4 genes 32 and 63 and was very close to the latter gene. It is, thus, in the region of the T4 map that is occupied by genes for a number of other enzymes, including deoxycytidylate deaminase, thymidylate synthetase, dihydrofolate reductase, and ribonucleotide reductase, that are nonessential to phage production in rich media.     

Functional Interaction of the tonA/tonB Receptor System in Escherichia coli

Host range mutants of phage T1 (T1h), which productively infected tonB mutants of Escherichia coli, were isolated. The phage mutants were inactivated by isolated outer membranes of E. coli in contrast to the wild-type phage, which only adsorbed reversibly. For the infection process, the tonB function is apparently only required for the irreversible adsorption of the phage T1, but not for the transfer of the phage DNA through the outer membrane and the cytoplasmic membrane of the cell. Mutants of the tonA gene expressing normal amounts of outer membrane receptor proteins were isolated and found to be partially sensitive to phage T5 and resistant to the phages T1 and T1h, colicin M, and albomycin and unable to take up iron as a ferrichrome complex. One tonA mutant remained partially sensitive to T5, colicin M, and albomycin and supported growth of T1h (not of T1) with the same plating efficiency as the parent strain. Only a small region of the tonA receptor protein seems to function for all the very different substrates. A newly isolated host range mutant of T5 (T5h) adsorbed faster to tonA(+) cells than did wild-type T5 and infected tonA missense mutants resistant to wild-type T5. The interplay of the tonA with the tonB function was observed with phage T5 infection, although T5 required only the tonA receptor. Ferrichrome inhibited plaque formation of T5 only when plated on tonB mutants. Adsorption of T5 to cells in liquid medium was influenced by ferrichrome as follows: complete inhibition by 0.1 muM ferrichrome with tonB mutants, not more than 35% inhibition by 1 to 100 muM ferrichrome with the tonB(+) parent strain in the presence of glucose as energy source, and 90% inhibition by 1 muM ferrichrome with partially starved parent cells. We conclude that there exist different functional states of the receptor protein that depend on the energy state of the cell and the tonB function. The latter seems to be required only for translocation processes with outer membrane proteins involved.     

Selective Allele Loss in Mixed Infections with T4 Bacteriophage

Evidence is presented that when E. coli B is mixedly infected with T4D wild type and rII deletion mutants, the excess DNA of the wild type allele is lost. No loss is seen in mixed infections with rII point mutants and wild type. In similar experiments with lysozyme addition mutants, the mutant allele is lost. We believe these results demonstrate a repair system which removes "loops" in heteroduplex DNA molecules. A number of phage and host functions have been tested for involvement in the repair of the excess DNA, and T4 genes x and v have been implicated in this process.