Properties of Bacteriophage T4 Mutants Defective in Gene 30 (Deoxyribonucleic Acid Ligase) and the rII Gene

In Escherichia coli K-12 strains infected with phage T4 which is defective in gene 30 [deoxyribonucleic acid (DNA) ligase] and in the rII gene (product unknown), near normal levels of DNA and viable phage were produced. Growth of such T4 ligase-rII double mutants was less efficient in E. coli B strains which show the "rapidlysis" phenotype of rII mutations. In pulse-chase experiments coupled with temperature shifts and with inhibition of DNA synthesis, it was observed that DNA synthesized by gene 30-defective phage is more susceptible to breakdown in vivo when the phage is carrying a wild-type rII gene. Breakdown was delayed or inhibited by continued DNA synthesis. Mutations of the rII gene decreased but did not completely abolish the breakdown. T4 ligase-rII double mutants had normal sensitivity to ultraviolet irradiation.     

Expression and DNA sequence of the cloned bacteriophage T4 dCMP hydroxymethylase gene.

A plasmid expressing the cloned bacteriophage T4 gene 42 gave the same levels of complementation of gene 42 mutants in a polarity-suppressing rho mutant as in a rho+ host. A reading frame likely corresponding to gene 42 and putative promoter and terminator sequences were identified in the partial sequence of the cloned fragment.     

Characterization of New Regulatory Mutants of Bacteriophage T4

Plating techniques which eliminate T4 plaque formation on Escherichia coli by folate analogue inhibition of dihydrofolate (FH(2)) reductase (EC 1.5.1.3) allowed the isolation of folate analogue-resistant (far) mutants of T4. One class of far mutants overproduces the phage-induced FH(2) reductase. Deoxycytidylate deaminase (EC 3.5.4.12), thymidine kinase (EC 2.7.1.21), and deoxycytidine triphosphatase (EC 3.6.1.12) are also overproduced by 20 min after infection at 37 C. The overproduction of FH(2) reductase by these far mutants is not affected by the absence of DNA synthesis. Other types of mutations that affect the synthesis of early enzymes cause overproduction in the absence of DNA synthesis of some of the above enzymes but not of FH(2) reductase. Therefore, overproducing far mutants apparently have mutations in previously undescribed genes controlling the expression of the T4 genome. Three of four mutants under study map near gene 56, and one maps near gene 52. All of these mutants show delays in DNA synthesis, phage production, and lysis and appear to show decreased levels of RNA synthesis based on the cumulative incorporation of uridine.     

Functional analysis of the phage T4 holin in a λ context

Phage lambda hybrids were constructed by inserting the t gene of phage T4 in place of the lambda holin gene, S. Induction of the hybrid phage resulted in lysis that was just as abrupt as, but occurred much earlier in the vegetative cycle than, that obtained with lambda, indicating that t is indeed a holin gene. Moreover, it was possible to impose lysis inhibition (LIN) on induction of the hybrid phage, but not of the parental lambda phage, by superinfection with LIN-competent T4. The imposition of the LIN state was found to depend on the allelic state of the rI and t genes of the superinfecting T4 phage, indicating that the LIN-sensitive state of the T holin is transient. Finally, induction of lysogens carrying both holin genes was shown to result in earlier triggering of lysis than with either holin gene alone. This result suggests that the two very dissimilar holins contribute additively to the physiology of the timing mechanism, or, less likely, that they interact to form one mass-action pool. In either case, these results imply a common pathway for holin timing and function.     

Lysis and lysis inhibition in bacteriophage T4: rV mutations reside in the holin t gene.

Upon infecting populations of susceptible host cells, T-even bacteriophages maximize their yield by switching from lysis at about 25 to 35 min at 37 degrees C after infection by a single phage particle to long-delayed lysis (lysis inhibition) under conditions of sequential infection occurring when free phages outnumber host cells. The timing of lysis depends upon gene t and upon one or more rapid-lysis (r) genes whose inactivation prevents lysis inhibition. t encodes a holin that mediates the movement of the T4 endolysin though the inner cell membrane to its target, the cell wall. The rI protein has been proposed to sense superinfection. Of the five reasonably well characterized r genes, only two, rI and rV, are clearly obligatory for lysis inhibition. We show here that rV mutations are alleles of t that probably render the t protein unable to respond to the lysis inhibition signal. The tr alleles cluster in the 5' third of t and produce a strong r phenotype, whereas conditional-lethal t alleles produce the classical t phenotype (inability to lyse) and other t alleles produce additional, still poorly understood phenotypes. tr mutations are dominant to t+, a result that suggests specific ways to probe T4 holin function.     

Candida albicans Rho-type GTPase-encoding genes required for polarized cell growth and cell separation

Rho proteins are essential regulators of morphogenesis in eukaryotic cells. In this report, we investigate the role of two previously uncharacterized Rho proteins, encoded by the Candida albicans RHO3 (CaRHO3) and CaCRL1/CaRHO4 genes. The CaRHO3 gene was found to contain one intron. Promoter shutdown experiments using a MET3 promoter-controlled RHO3 revealed a strong cell polarity defect and a partially depolarized actin cytoskeleton. Hyphal growth after promoter shutdown was abolished in rho3 mutants even in the presence of a constitutively active ras1(G13V) allele, and existing germ tubes became swollen. Deletion of C. albicans RHO4 indicated that it is a nonessential gene and that rho4 mutants were phenotypically different from rho3. Two distinct phenotypes of rho4 cells were elongated cell morphology and an unexpected cell separation defect generating chains of cells. Colony morphology of crl1/rho4 resulted in a growth-dependent smooth (long cell cycle length) or wrinkled (short cell cycle length) phenotype. This phenotype was additionally dependent on the rho4 cell separation defect and was also found in a Cacht3 chitinase mutant that shows a strong cytokinesis defect. The overexpression of the endoglucanase encoding the ENG1 gene, but not CHT3, suppressed the cell separation defect of crl1/rho4 but could not suppress the cell elongation phenotype. C. albicans Crl1/Rho4 and Bnr1 both localize to septal sites in yeast and hyphal cells but not to the hyphal tip. Deletion of RHO4 and BNR1 produced similar morphological phenotypes. Based on the localization of Rho4 and on the rho4 mutant phenotype, we propose a model in which Rho4p may function as a regulator of cell polarity, breaking the initial axis of polarity found during early bud growth to promote the construction of a septum.     

Heat-inducible mutants of corynebacteriophage.

Heat-inducible mutants of temperate cornebacteriophage beta and gamma, called temperature-sensitive repression (tsr) mutants, were isolated and characterized. Lysogens carrying these mutants were induced at 38 degrees C, produced a normal or slightly increased yield of phage, and underwent extensive lysis at this temperature. In some cases mutation to heat inducibility had altered the UV inducibility of the phage, the changes ranging from loss to enhancement of this trait. Complementation tests showed that all five beta-tsr strains had mutated in the same cistron and suggested that these mutations were in the gene responsible for repressor production.     

Lysis of lysis-inhibited bacteriophage T4-infected cells.

T4 bacteriophage (phage)-infected cells show a marked increase in latent-period length, called lysis inhibition, upon adsorption of additional T4 phages (secondary adsorption). Lysis inhibition is a complex phenotype requiring the activity of at least six T4 genes. Two basic mysteries surround our understanding of the expression of lysis inhibition: (i) the mechanism of initiation (i.e., how secondary adsorption leads to the expression of lysis inhibition) and (ii) the mechanism of lysis (i.e., how this signal not to lyse is reversed). This study first covers the basic biology of the expression of lysis inhibition and lysis of T4-infected cells at high culture densities. Then evidence is presented which implies that, as with the initiation of lysis inhibition, sudden, lysis-associated clearing of these cultures is likely caused by T4 secondary adsorption. For example, such clearing is often observed for lysis-inhibited T4-infected cells grown in batch culture during T4 stock preparation. The significance of this secondary adsorption-induced lysis to wild T4 populations is discussed. The study concludes with a logical argument suggesting that the lytic nature of the T4 phage particle evolved as a novel mechanism of phage-induced lysis.     

Selection of lambda Spi- transducing phages using the P2 old gene cloned onto a plasmid

The old gene product of the P2 prophage interferes with plaque formation by lambda wild type phage but allows lambda phages whose red and gam genes have been deleted to form small, visible plaques (the lambda Spi- phenotype). The old gene product also kills Escherichia coli recB or recC mutants. We have cloned the old gene into the high-copy-number plasmid pBR322, where it prevents plaque formation by both lambda Spi+ and lambda Spi- phages. We transferred a DNA fragment that carries the old gene to the low-copy-number plasmid pSC101 and found that lambda Spi- phages can be selected on strains that carry this plasmid. The plasmid-borne old gene kills E. coli recB mutants, providing a selection for old- mutants.     

Fission yeast Rho5p GTPase is a functional paralogue of Rho1p that plays a role in survival of spores and stationary-phase cells

The Rho GTPase family and their effectors are key regulators involved in many eukaryotic cell functions related to actin organization and polarity establishment. Schizosaccharomyces pombe Rho1p is essential, directly activates the (1,3)-beta-d-glucan synthase, and participates in regulation of cell wall growth and morphogenesis. Here we describe the characterization of the fission yeast Rho5p GTPase, highly homologous to Rho1p, sharing 86% identity and 95% similarity. Overexpression of the hyperactive allele rho5-G15V causes a morphological effect similar to that of rho1-G15V, but the penetrance is significantly lower, and overexpression of the dominant-negative allele rho5-T20N causes lysis like that of rho1-T20N. Importantly, overexpression of rho5(+) but no other rho genes is able to rescue the lethality of rho1Delta cells. Shutoff experiments indicated that Rho5p can replace Rho1p, but it is not as effective in maintaining cell wall integrity or actin organization. rho5(+) expression is hardly detected during log-phase growth but is induced under nutritional starvation conditions. rho5Delta cells are viable and do not display any defects during logarithmic growth. However, when rho1(+) expression is repressed during stationary phase, rho5Delta cells display reduced viability. Ascospores lacking Rho5p are less resistant to heat or lytic enzymes than wild-type spores. Moreover, h(90) mutant strains carrying the hyperactive rho5-G15V or the dominant-negative rho5-T20N alleles display severe ascospore formation defects. These results suggest that Rho5p functions in a way similar to, but less efficient than, Rho1p, plays a nonessential role during stationary phase, and participates in the spore wall formation.     

P2 growth restriction on an rpoC mutant is suppressed by alleles of the Rz1 homolog lysC

Escherichia coli strain 397c carries a temperature-sensitive mutation, rpoC397, that removes the last 50 amino acids of the RNA polymerase beta' subunit and is nonpermissive for plating of bacteriophage P2. P2 gor mutants productively infect 397c and define a new gene, lysC, encoded by a reading frame that extensively overlaps the P2 lysis accessory gene, lysB. The unusual location of lysC with respect to lysB is reminiscent of the Rz/Rz1 lysis gene pair of phage lambda. Indeed, coexpression of lysB and lysC complemented the growth defect of lambda Rz/Rz1 null mutants, indicating that the LysB/C pair is similar to Rz/Rz1 in both gene arrangement and function. Cells carrying the rpoC397 mutation exhibited an early onset of P2-induced lysis, which was suppressed by the gor mutation in lysC. We propose that changes in host gene expression resulting from the rpoC397 mutation result in changes in the composition of the bacterial cell wall, making the cell more susceptible to P2-mediated lysis and preventing accumulation of progeny phage sufficient for plaque formation.     

The Escherichia coli FKBP-type PPIase SlyD is required for the stabilization of the E lysis protein of bacteriophage phi X174

Most bacteriophages abruptly terminate their vegetative cycle by causing lysis of the host cell. The ssDNA phage phi X174 uses a single lysis gene, E, encoding a 91-amino-acid membrane protein that causes lysis of Escherichia coli by inhibiting MraY, a conserved enzyme of murein biosynthesis. Recessive mutations in the host gene slyD (sensitivity to lysis) absolutely block E-mediated lysis and phi X174 plaque formation. The slyD gene encodes a FKBP-type peptidyl-prolyl cis-trans isomerase (PPIase). To investigate the molecular basis of this unique FKBP-dependence, spontaneous plaque-forming mutants of phi X174 were isolated on a slyD lawn. All of these Epos ('plates on slyD') suppressors encode proteins with either a R3H or L19F change. The double mutant was also isolated and generated the largest plaques on the slyD lawn. A c-myc epitope tag sequence was incorporated into the parental E and Epos genes without effect on lytic function. Western blots and pulse-chase labelling experiments showed that both Epos and E are highly unstable in a slyD background; however, Epos is synthesized at a higher rate, allowing a lysis-sufficient level of Epos to accumulate. Our results indicate that SlyD is required for stabilizing the E protein and allowing it to accumulate to the levels required to exert its lytic effect. These data are discussed in terms of a model for the specific role of the SlyD PPIase in E folding, and of the use of the very strict SlyD- dependence phenotype for identifying elements of PPIase selectivity.     

Evolution of bacteriophage T7 in a growing plaque.

The emergence of mutants during the 10(9)-fold amplification of a bacteriophage was spatially resolved in a growing plaque. When wild-type phage T7 was grown on an Escherichia coli host which expressed an essential early enzyme of the phage infection cycle, the T7 RNA polymerase, mutant phage relying on this enzyme appeared in 10(8) phage replications and outgrew the wild type. Spatial resolution of the selection process was achieved by analyzing stab samples taken along a plaque radius. Different mutants were selected at different rates along different radii of the plaque, based on host range and restriction patterns of the isolates. The mutants deleted up to 11% of their genomes, including the gene for their own RNA polymerase. They gained an advantage over the wild type by replicating more efficiently, as determined by one-step growth cultures.     

Chromogenic detection of antigen in bacteriophage plaques: a microplaque method applicable to large-scale screening

We have developed a simple and rapid (24 h) enzyme-linked immuno-detection method to screen for rare antigen-positive phage among large numbers of antigen-negative ones. Horse-radish peroxidase-antibody conjugate, incorporated into the soft agar layer of a plaque assay system, is precipitated locally by antigen produced during plaque formation, and is detected by standard chromogenic methods. The method has been used to screen plaques of bacteriophage beta tox+ for the presence of diphtheria toxin and related cross-reacting material. When phage were plated on very dense bacterial lawns, they formed minute plaques (microplaques). Because of the high local concentration of antigen generated by lysis of the dense lawn, the microplaques gave more intense chromogenic signals than larger plaques formed on less dense Corynebacterium diphtheriae lawns. Thus, antigen-positive microplaques could be easily recognized even in the presence of very large numbers of antigen-negatives. In a reconstruction experiment, small numbers of antigen-positive phage were detected with high efficiency (greater than 75%) against a background of 3.8 X 10(4) antigen-negatives/cm2 of agar surface (equivalent to 2.4 X 10(6) plaques/9 cm petri plate). This screening method should facilitate isolation of phage mutants affecting production of given antigens and may be of particular value in detecting specific genes cloned into phage vectors.     

A Rho-type GTPase, rho-4, is required for septation in Neurospora crassa

Proteins in the Rho family are small monomeric GTPases primarily involved in polarization, control of cell division, and reorganization of cytoskeletal elements. Phylogenetic analysis of predicted fungal Rho proteins suggests that a new Rho-type GTPase family, whose founding member is Rho4 from the archiascomycete Schizosaccharomyces pombe, is involved in septation. S. pombe rho4Delta mutants have multiple, abnormal septa. In contrast to S. pombe rho4Delta mutants, we show that strains containing rho-4 loss-of-function mutations in the filamentous fungus Neurospora crassa lead to a loss of septation. Epitope-tagged RHO-4 localized to septa and to the plasma membrane. In other fungi, the steps required for septation include formin, septin, and actin localization followed by cell wall synthesis and the completion of septation. rho-4 mutants were unable to form actin rings, showing that RHO-4 is required for actin ring formation. Characterization of strains containing activated alleles of rho-4 showed that RHO-4-GTP is likely to initiate new septum formation in N. crassa.     

Lysis gene t of T-even bacteriophages: evidence that colicins and bacteriophage genes have common ancestors.

The lysis gene t of the T-even-like bacteriophage K3 has been cloned and sequenced. The gene codes for a protein with a predicted molecular weight of 25,200. Expression of the complete lysis protein was impossible, but peptides complementing T4 amber mutants in t are described. No known lysis protein of other phages is homologous to protein T. Also, the Escherichia coli phospholipase A is different from protein T. CelB, the lysis protein of the colicin E2 operon, shows a similarity to protein T. Sequences of colicins A, E1, and E2 are related to gene 38 sequences, the gene preceding t and coding for the phage adhesin. A common origin for colicin genes and phage genes is discussed, and a protein region in colicins that is responsible for receptor recognition is predicted.     

Functional Interactions between the DNA Ligase of ESCHERICHIA COLI and Components of the DNA Metabolic Apparatus of T4 Bacteriophage

T4 phage completely defective in both gene 30 (DNA ligase) and the rII gene (function unknown) require at least normal levels of host-derived DNA ligase (E. coli lig gene) for growth. Viable E. coli mutant strains that harbor less than 5% of the wild-type level of bacterial ligase do not support growth of T4 doubly defective in genes 30 and rII (T4 30- rII- mutants). We describe here two classes of secondary phage mutations that permit the growth of T4 30- rII- phage on ligase-defective hosts. One class mapped in T4 gene su30 (Krylov 1972) and improved T4 30- rII- phage growth on all E. coli strains, but to varying degrees that depended on levels of residual host ligase. Another class mapped in T4 gene 32 (helix-destabilizing protein) and improved growth specifically on a host carrying the lig2 mutation, but not on a host carrying another lig- lesion (lig4). Two conclusions are drawn from the work: (1) the role of DNA ligase in essential DNA metabolic processes in T4-infected E. coli is catalytic rather than stoichiometric, and (2) the E. coli DNA ligase is capable of specific functional interactions with components of the T4 DNA replication and/or repair apparatus.     

Phage t: a group T plasmid-dependent bacteriophage

Phage t was isolated from sewage from Pretoria. It formed plaques only on Escherichia coli and Salmonella typhimurium strains that carried plasmids belonging to incompatibility group T. Five of six group T plasmids permitted visible lysis of R+ host strains. There was no visible lysis of E. coli J53-2 or S. typhimurium LT2trpA8 carrying the T plasmid Rts1 although the strains supported phage growth as indicated by at least a 10-fold increase in phage titre. The latter strains transferred the plasmid at high frequency to E. coli strain CSH2 and the resulting transconjugants plated the phage. Proteus mirabilis strain PM5006(R402) failed to support phage growth although it transferred the plasmid and concomitant phage sensitivity to E. coli J53-2. The phage was hexagonal in outline, RNA-containing, resistant to chloroform and adsorbed to the shafts of pili determined by T plasmids.