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R Calendar

Publications and source records attributed to R Calendar.

At least 37 records · Page 2Linked to original sources
PubMed1995

The old exonuclease of bacteriophage P2.

The Old protein of bacteriophage P2 is responsible for interference with the growth of phage lambda and for killing of recBC mutant Escherichia coli. We have purified Old fused to the maltose-binding protein to 95% purity and characterized its enzymatic properties. The Old protein fused to maltose-binding protein has exonuclease activity on double-stranded DNA as well as nuclease activity on single-stranded DNA and RNA. The direction of digestion of double-stranded DNA is from 5' to 3', and digestion initiates at either the 5'-phosphoryl or 5'-hydroxyl terminus. The nuclease is active on nicked circular DNA, degrades DNA in a processive manner, and releases 5'-phosphoryl mononucleotides.

Adenosine Triphosphatases↗
PubMed1994

Molecular cloning and characterization of bacteriophage P2 genes R and S involved in tail completion.

The sequences of two previously known tail genes, R and S, of the temperate bacteriophage P2 and the sequence of an additional open reading frame (orf-30) located between S and V, were determined. Amber mutations mapping within R and S, Ram3, Ram42, Ram23, Sam75, and Sam89 were sequenced and found to be within their corresponding open reading frames. We constructed overproducing plasmids for R and S and identified these proteins by SDS-PAGE of whole-cell lysates and Coomassie blue staining. The predicted molecular masses of proteins R and S were M(r) 17,400 and 17,300, respectively, although both polypeptides migrated more slowly during gel electrophoresis than would be expected from the sequence data. orf-30 occupies the strand opposite from RS and V and is preceded by several weak potential sigma 70-RNA polymerase promoters, some of which overlap with the V promoter. A construct that had the putative orf-30 promoter region upstream of the lacZ gene produced low levels of beta-galactosidase activity in vivo. Expression from the orf-30 promoter was not stimulated by the phage P4 transcriptional activator protein, delta, which acts at all the known P2 and P4 late promoters. Insertion mutagenesis showed that orf-30 was not an essential gene for P2 growth in Escherichia coli. None of the gene or protein sequences exhibited extensive homology to sequences in the nucleic acid and protein databases. However, the R protein contains a small region homologous to one in the phage T4 tail protein gp15, which is required for T4 tails to bind heads. We propose that R and S are tail completion proteins that are essential for stable head joining.

Amino Acid Sequence↗
PubMed1994

Functions involved in bacteriophage P2-induced host cell lysis and identification of a new tail gene.

Successful completion of the bacteriophage P2 lytic cycle requires phage-induced lysis of its Escherichia coli host, a process that is poorly understood. Genetic analysis of lysis-deficient mutants defined a single locus, gene K, which lies within the largest late transcription unit of P2 and maps between head gene L and tail gene R. We determined and analyzed the DNA sequence of a ca. 2.1-kb EcoRV fragment that spans the entire region from L to R, thus completing the sequence of this operon. This region contains all of the functions necessary for host cell lysis. Sequence analysis revealed five open reading frames, initially designated orf19 through orf23. All of the existing lysis mutants--ts60, am12, am76, and am218--were located in orf21, which must therefore correspond to gene K. The K gene product has extensive amino acid sequence similarity to the product of gene R of bacteriophage lambda, and its exhibits endolysin function. Site-directed mutagenesis and reverse genetics were used to create P2 amber mutants in each of the four other newly identified open reading frames. Both orf19 (gene X) and orf20 (gene Y) encode essential functions, whereas orf22 (lysA) and orf23 (lysB) are nonessential. Gene Y encodes a polypeptide with striking similarities to the family of holin proteins exemplified by gpS of phage lambda, and the Yam mutant displayed the expected properties of a holin mutant. The gene products of lysA and lysB, although nonessential, appear to play a role in the correct timing of lysis, since a lysA amber mutant caused slightly accelerated lysis and a lysB amber mutant slightly delayed lysis of nonpermissive strains. Gene X must encode a tail protein, since lysates from nonpermissive cells infected with the X amber mutant were complemented in vitro by similar lysates of cells infected with P2 head mutants but not with tail mutants.

Amino Acid Sequence↗
PubMed1994

A new gene of bacteriophage P4 that controls DNA replication.

Bacteriophage P4 replication may result in either a lytic cycle or plasmid maintenance, depending on the presence or absence, respectively, of helper phase P2 genome. Bacteriophage P4 DNA replication depends on the product of gene alpha, which has origin recognition, primase, and helicase activities. An open reading frame with the coding capacity for a protein of 106 amino acids (orf106) is located upstream of the alpha gene. Genes orf106 and alpha are transcriptionally coregulated. Three amber mutations and an internal deletion (del51) were introduced into orf106. All of the amber mutations exhibited a polar effect on transcription of the downstream alpha gene. The P4 del51 mutant was slightly defective in lytic growth and could not be propagated in the plasmid state. In this latter condition, P4 DNA overreplication was observed. Overexpression of Orf106 severely inhibited P4 DNA replication, preventing P4 lytic growth and plasmid maintenance. The inhibitory effect of Orf106 on P4 replication was not observed when both orf106 and alpha were overexpressed. We suggest that orf106 is involved in P4 replication and that a balanced expression of orf106 relative to alpha may be necessary for proper P4 DNA replication. In particular, orf106 appears to be essential for the control of P4 genome replication in the plasmid state. We propose that orf106 be named cnr, for copy number regulation.

Amino Acid Sequence↗
PubMed1993

Mechanisms of genome propagation and helper exploitation by satellite phage P4.

Temperate coliphage P2 and satellite phage P4 have icosahedral capsids and contractile tails with side tail fibers. Because P4 requires all the capsid, tail, and lysis genes (late genes) of P2, the genomes of these phages are in constant communication during P4 development. The P4 genome (11,624 bp) and the P2 genome (33.8 kb) share homologous cos sites of 55 bp which are essential for generating 19-bp cohesive ends but are otherwise dissimilar. P4 turns on the expression of helper phage late genes by two mechanisms: derepression of P2 prophage and transactivation of P2 late-gene promoters. P4 also exploits the morphopoietic pathway of P2 by controlling the capsid size to fit its smaller genome. The P4 sid gene product is responsible for capsid size determination, and the P2 capsid gene product, gpN, is used to build both sizes. The P2 capsid contains 420 capsid protein subunits, and P4 contains 240 subunits. The size reduction appears to involve a major change of the whole hexamer complex. The P4 particles are less stable to heat inactivation, unless their capsids are coated with a P4-encoded decoration protein (the psu gene product). P4 uses a small RNA molecule as its immunity factor. Expression of P4 replication functions is prevented by premature transcription termination effected by this small RNA molecule, which contains a sequence that is complementary to a sequence in the transcript that it terminates.

Amino Acid Sequence↗
PubMed1992

A common sequence motif, -E-G-Y-A-T-A-, identified within the primase domains of plasmid-encoded I- and P-type DNA primases and the alpha protein of the Escherichia coli satellite phage P4.

DNA primases encoded by the conjugative plasmids ColIb-P9 (IncI1), RP4, and R751 (IncP), and the protein of the Escherichia coli satellite phage P4 alpha were shown to contain a common amino acid sequence motif -E-G-Y-A-T-A-. The P4 alpha gene product, required for initiation of phage DNA replication, exhibits primase activity on single-stranded circular DNA templates. This priming activity resembles the enzymatic activity of DNA primases encoded by conjugative plasmids in terms of template utilization and the ability to synthesize primers that can be elongated by DNA polymerase III holoenzyme. The -E-G-Y-A-T-A- motif is part of an extended sequence region most conserved within the primase domains of the four enzymes. Single amino acid substitutions generated in the -E-G-Y-A-T-A- motif of the RP4 TraC2 and the P4 alpha protein affect priming activity, supporting the hypothesis that the conserved sequence motif is part of the active center for primase function. A mutation that eliminates priming activity causes P4 phage to grow poorly and to depend upon the host dnaG primase. Computer analysis identified two additional sequence motifs within the amino acid sequence of the P4 alpha protein: a potential zinc-finger motif and a "type A" nucleotide binding site, both strikingly similar to sequence motifs described in various DNA primases and helicases.

Amino Acid Sequence↗
PubMed1992

The polarity suppression factor of bacteriophage P4 is also a decoration protein of the P4 capsid.

We show that the product of the polarity suppression (psu) gene from bacteriophage P4 associates with P4 capsids. This association can occur when Psu is (i) provided in vivo from the P4 genome or from a plasmid or (ii) provided in vitro by mixing viable phage particles with Psu protein. Psu is unable to associate with the larger capsid of P4's helper phage P2. Discrimination of the P4 and P2 capsids by Psu appears to be independent of the presence of the P4 genome in the capsid, since P2 size capsids filled with P4 DNA cannot accommodate Psu association. P4 psu particles devoid of Psu are less stable than P4 particles carrying Psu. These results indicate that, in addition to its antitermination activity at Rho-dependent terminators, Psu is also a decoration protein that stabilizes the P4 capsids.

Amino Acid Sequence↗
PubMed1992

DNA sequences of the tail fiber genes of bacteriophage P2: evidence for horizontal transfer of tail fiber genes among unrelated bacteriophages.

We have determined the DNA sequence of the bacteriophage P2 tail genes G and H, which code for polypeptides of 175 and 669 residues, respectively. Gene H probably codes for the distal part of the P2 tail fiber, since the deduced sequence of its product contains regions similar to tail fiber proteins from phages Mu, P1, lambda, K3, and T2. The similarities of the carboxy-terminal portions of the P2, Mu, ann P1 tail fiber proteins may explain the observation that these phages in general have the same host range. The P2 H gene product is similar to the products of both lambda open reading frame (ORF) 401 (stf, side tail fiber) and its downstream ORF, ORF 314. If 1 bp is inserted near the end of ORF 401, this reading frame becomes fused with ORF 314, creating an ORF that may represent the complete stf gene that encodes a 774-amino-acid-long side tail fiber protein. Thus, a frameshift mutation seems to be present in the common laboratory strain of lambda. Gene G of P2 probably codes for a protein required for assembly of the tail fibers of the virion. The entire G gene product is very similar to the products of genes U and U' of phage Mu; a region of these proteins is also found in the tail fiber assembly proteins of phages TuIa, TuIb, T4, and lambda. The similarities in the tail fiber genes of phages of different families provide evidence that illegitimate recombination occurs at previously unappreciated levels and that phages are taking advantage of the gene pool available to them to alter their host ranges under selective pressures.

Amino Acid Sequence↗
PubMed1991

Nucleotide sequence of the genes encoding the major tail sheath and tail tube proteins of bacteriophage P2.

The major structural components of the contractile tail of bacteriophage P2 are proteins FI and FII, which are believed to be the tail sheath and tube proteins, respectively. Both proteins were mapped previously to the P2 late gene F, based on the pattern of protein synthesis in various P2 amber mutants. In order to clarify the gene arrangement and to provide a basis for structural comparisons with other contractile phage tails, we have determined the nucleotide sequence of the region of the P2 genome encoding these two proteins. The coding regions were confirmed by location of the Fam4 mutation and by N-terminal amino acid sequencing of both proteins. The molecular weight and amino acid composition predicted by each of the coding regions correspond well to those determined experimentally for each protein. FII is encoded by a newly identified P2 late gene. These proteins bear little resemblance to their functional homologues in bacteriophage T4.

Amino Acid Sequence↗
PubMed1991

Nucleotide sequence of the DNA packaging and capsid synthesis genes of bacteriophage P2.

Overlapping DNA fragments containing the DNA packaging and capsid synthesis gene region of bacteriophage P2 were cloned and sequenced. In this report we present the complete nucleotide sequence of this 6550 bp region. Each of six open reading frames found in the interval was assigned to one of the essential genes (Q, P, O, N, M and L) by correlating genetic, physical and mutational data with DNA and protein sequence information. Polypeptides predicted were: a capsid completion protein, gpL; the major capsid precursor, gpN; the presumed capsid scaffolding protein; gpO; the ATPase and proposed endonuclease subunits of terminase, gpP and gpM, respectively; and a candidate for the portal protein, gpQ. These gene and protein sequences exhibited no homology to analogous genes or proteins of other bacteriophages. Expression of gene Q in E. coli from a plasmid caused production of a Mr 39,000 Da protein that restored Qam34 growth. This sequence analysis found only genes previously known from analysis of conditional-lethal mutations. No new capsid genes were found.

Adenosine Triphosphatases↗
PubMed1991

Mutational analysis of a bacteriophage P4 late promoter.

Transcription from the late Psid promoter of satellite bacteriophage P4 is dependent on the bacterial RNA polymerase carrying the sigma 70 subunit and is positively regulated by the product of the P4 delta gene or the ogr gene of helper bacteriophage P2. Through deletion and mutational analyses of the Psid promoter, we identified mutations in the -10 region and in a region of hyphenated dyad symmetry centered around position -55 that inactivate Psid. Most of these mutations alter base pairs that are highly conserved in the five other delta-activated P4 and P2 late promoters. We propose that the P4 delta and P2 ogr gene products bind the -55 region of the P4 and P2 late promoters.

Base Sequence↗
PubMed1990

Characterization of the cos sites of bacteriophages P2 and P4.

A 641-bp cos-containing P2 DNA fragment was sequenced and compared to the P4 cos region. Alignment of the P2 and P4 cos regions shows a homologous region of 55 bp that has only three mismatches and contains a completely conserved region of dyad symmetry. A number of P4- and P2-derived cosmids were tested in an in vivo transduction assay in order to determine the minimal cos region required for packaging. These experiments show that the common region of 55 bp is sufficient for transduction with low frequency, but that a 125-bp cos-containing fragment contains all the information for transduction with optimal frequency.

Bacteriophages↗
PubMed1990

A mutation of the transactivation gene of satellite bacteriophage P4 that suppresses the rpoA109 mutation of Escherichia coli.

Satellite bacteriophage P4 requires the products of the late genes of a helper such as P2 in order to grow lytically. The Escherichia coli rpoA109 mutation, which alters the alpha subunit of RNA polymerase, prevents transcription of the late genes of bacteriophage P2. Suppressor mutations that define the P2 ogr gene overcome this block. We found that P4 lytic growth using a P2 ogr+ prophage helper was prevented by the rpoA109 mutation but that this block was overcome when the P2 helper carried the suppressor mutation in the ogr gene. Furthermore, we isolated and characterized four independent mutations in P4, called org, that suppress the E. coli rpoA109 mutation by allowing P4 lytic growth using a P2 ogr+ helper. DNA sequence analysis revealed that the four independent org mutations are identical and that they occur in the P4 delta gene, which codes for a factor that positively regulates the transcription of the P2 and P4 late genes. delta is predicted to code for a basic 166-amino-acid residue protein. Each 83-residue half of the predicted delta gene product is similar to the predicted 72-residue proteins encoded by the ogr gene of P2 and the B gene of phage 186.

Alleles↗
PubMed1990

Bacteriophage P2 ogr and P4 delta genes act independently and are essential for P4 multiplication.

Satellite bacteriophage P4 requires the products of the late genes of a helper phage such as P2 for lytic growth. Expression of the P2 late genes is positively regulated by the P2 ogr gene in a process requiring P2 DNA replication. Transactivation of P2 late gene expression by P4 requires the P4 delta gene product and works even in the absence of P2 DNA replication. We have made null mutants of the P2 ogr and P4 delta genes. In the absence of the P4 delta gene product, P4 multiplication required both the P2 ogr protein and P2 DNA replication. In the absence of the P2 ogr gene product, P4 multiplication required the P4 delta protein. In complementation experiments, we found that the P2 ogr protein was made in the absence of P2 DNA replication but could not function unless P2 DNA replicated. We produced P4 delta protein from a plasmid and found that it complemented the null P4 delta and P2 ogr mutants.

Amino Acid Sequence↗
PubMed1990

Nonessential region of bacteriophage P4: DNA sequence, transcription, gene products, and functions.

We sequenced the leftmost 2,640 base pairs of bacteriophage P4 DNA, thus completing the sequence of the 11,627-base-pair P4 genome. The newly sequenced region encodes three nonessential genes, which are called gop, beta, and cII (in order, from left to right). The gop gene product kills Escherichia coli when the beta protein is absent; the gop and beta genes are transcribed rightward from the same promoter. The cII gene is transcribed leftward to a rho-independent terminator. Mutation of this terminator creates a temperature-sensitive phenotype, presumably owing to a defect in expression of the beta gene.

Alleles↗
PubMed1990

Interactions between satellite bacteriophage P4 and its helpers.

The helper dependence of satellite phage P4 superimposes an additional set of regulatory interactions on those required for the independent maintenance of P4 or its helpers. These interactions allow P4 to exploit a helper phage under a variety of circumstances and can affect expression of the immunity functions and late genes of both phages. The phage P2 lysis/lysogeny decision involves two competing repressors regulating mutually exclusive promoters in the early control region. In the absence of a helper phage, the P4 immunity function plays a role in the choice between lysogeny or the multicopy plasmid state. No evidence exists for a P4-encoded immunity repressor; in P4-lysogenic cells, expression of the P4 DNA replication gene alpha appears to be prevented by premature termination of transcription. Immunity-independent expression of alpha in the multicopy plasmid state involves initiation of transcription at an alternative upstream promoter that is positively regulated by P4 delta protein; the same promoter is activated by P2 Cox protein during derepression of P4 by P2. The mechanism of derepression of P2 by P4 remains to be determined, and the relationship between the P4 immunity and derepression functions and the mutations that allow P4 to grow with a P3 prophage helper is an intriguing area for further exploration. Expression of P2 and P4 late genes is regulated by phage-encoded, zinc-binding transcriptional activators that appear to interact directly with the alpha subunit of RNA polymerase of E. coli. Stimulation of P2 late transcription by P2 Ogr protein depends upon phage DNA replication, whereas activation of transcription from the same promoters by the related P4 delta gene product is replication-independent. Elucidation of the mechanisms underlying these interactions promises to provide new insights into strategies for control of gene expression.

Amino Acid Sequence↗