The complete genomes of Staphylococcus aureus bacteriophages 80 and 80α--implications for the specificity of SaPI mobilization.

Staphylococcus aureus pathogenicity islands (SaPIs) are mobile elements that are induced by a helper bacteriophage to excise and replicate and to be encapsidated in phage-like particles smaller than those of the helper, leading to high-frequency transfer. SaPI mobilization is helper phage specific; only certain SaPIs can be mobilized by a particular helper phage. Staphylococcal phage 80α can mobilize every SaPI tested thus far, including SaPI1, SaPI2 and SaPIbov1. Phage 80, on the other hand, cannot mobilize SaPI1, and ϕ11 mobilizes only SaPIbov1. In order to better understand the relationship between SaPIs and their helper phages, the genomes of phages 80 and 80α were sequenced, compared with other staphylococcal phage genomes, and analyzed for unique features that may be involved in SaPI mobilization.

Mutational analysis of a satellite phage activator.

The late gene activator, Delta, of satellite phage P4 is more efficient than the Delta of satellite phage phiR73 in utilizing a P2 helper prophage that lacks an activator (ogr) gene. Analysis of P4 Delta is complicated by the fact that this protein contains two tandem phiR73 Delta-like domains. We performed a mutational analysis of phiR73 Delta, in order to select mutations that might not be found using P4 Delta. The host RNA polymerase alpha subunit mutation rpoA155 (L289F) blocks the growth of P2, P4, and P4 carrying the delta gene of phiR73. A mutant of this latter phage that can grow in the presence of rpoA155 carries a V19M mutation in phiR73 Delta. This suggests that aa 19 contacts RNA polymerase, in addition to the aa residues 13, 42 and 44, that have been implicated in interactions with RNA polymerase by previous mutational analyses of P2 ogr and P4 delta. In corroboration of the proposed role of the regions at aa residues 19, 42, and 44, we found phiR73 Delta mutations in these regions that showed a reduced activation of late gene expression, but a normal ability to bind to late gene promoters. All activators in the Delta class contain four Cys residues that bind Zn2+. Mutation of these aa residues in phiR73 Delta eliminated late gene activation. Spectroscopic analysis of these mutant proteins revealed that they were unable to bind Zn2+. Histidine residues were substituted for two of the Cys residues (C30 and C35), changing a C2C2 type Zn-binding motif to a C2H2 motif. Although His residues are used in coordinating Zn2+ in other proteins, these His substitutions resulted in complete loss of activity and the inability to bind Zn2+.

An upstream sequence element required for NucC-dependent expression of the Serratia marcescens extracellular nuclease.

The Serratia marcescens extracellular nuclease gene, nucA, is positively regulated by the product of the nucC gene. In this study, the upstream region required for NucC-dependent nuclease expression was defined by using fusions to the gene encoding chloramphenicol acetyltransferase (cat). This sequence includes an element of hyphenated dyad symmetry identified previously as the binding site for the P2 Ogr family of activators. Footprint analysis confirmed that members of this family of activator proteins bind to this site, protecting a region between -76 and -59 relative to the start of transcription. The activator binding site in the nucA promoter lies one turn of the helix upstream from the corresponding sites in the P2 and P4 late promoters. The effects of deletions between the downstream end of the activator binding site and the putative -35 region are consistent with a strict helical phasing requirement for activation.

Activation of P2 late transcription by P2 Ogr protein requires a discrete contact site on the C terminus of the alpha subunit of Escherichia coli RNA polymerase.

Bacteriophage P2 late transcription requires the product of the P2 ogr gene. Ogr-dependent transcription from P2 late promoters is blocked by certain point mutations affecting the alpha subunits of the host RNA polymerase. An alanine scan spanning the putative activation target in the alpha C-terminal domain (alphaCTD) was carried out to identify individual residues essential for Ogr-dependent transcription from P2 late promoters. In addition, the effects of alanine substitutions in the regions of the alphaCTD previously reported to affect CAP-dependent activation of the lac promoter and UP-element DNA binding were examined. Residues E286, V287, L289 and L290 in helix 3, and residue L300 at the beginning of helix 4, define a surface-exposed patch on the alphaCTD important for Ogr-dependent activation. These residues, adjacent to the recently identified DNA-binding determinants, constitute a newly identified activation surface for protein:protein contact. Alanine substitutions at some of the residues that affect UP-element DNA binding also impaired activation. This suggests that upstream DNA-alpha contacts, in addition to alpha-Ogr contacts, may be important in P2 late transcription. Other residues implicated in the interaction of alpha with CAP are not required for activation by Ogr, consistent with previous genetic evidence suggesting that these activators contact different sites on the alphaCTD.

Escherichia coli rpoC397 encodes a temperature-sensitive C-terminal frameshift in the beta' subunit of RNA polymerase that blocks growth of bacteriophage P2.

Escherichia coli 397c is temperature sensitive for growth at 43.5 degrees C and unable to plate bacteriophage P2 at 33 degrees C. The mutation conferring these phenotypes was mapped to the rpoC gene. RNA synthesis is temperature sensitive in the mutant strain, and the beta' subunit of RNA polymerase isolated from this strain exhibits increased electrophoretic mobility. DNA sequence analysis revealed that the mutation is a deletion of 16 bp, resulting in a frameshift that leads to truncation of the beta' subunit at the carboxy terminus.

Regulation of the Serratia marcescens extracellular nuclease: positive control by a homolog of P2 Ogr encoded by a cryptic prophage.

The Serratia marcescens extracellular nuclease is a secreted protein that is subject to growth phase and SOS control. Regulatory mutants affecting nuclease expression have been isolated that define a new locus, nucC, essential for transcription of the nuclease gene nucA. The cloned nucC gene is able to activate efficient expression from the nucA promoter in Escherichia coli, where it normally is poorly expressed. NucC is very similar to the bacteriophage P2 Ogr protein, a transcriptional activator essential for P2 late gene expression. NucC is able to replace P2 Ogr to support the growth of P2 ogr- mutants in E. coli. Ogr is a poor activator of the nuclease promoter in E. coli, but the related delta gene product from satellite phage P4 is highly effective. The presence of genes encoding a lysozyme and a putative porin or holin in the nucC operon suggests that nucC may be part of a cryptic prophage genome. The putative holin-like membrane protein is required in E. coli for extracellular secretion of the S. marcescens nuclease.

The sigma-70 subunit from Escherichia coli C differs from that of E. coli K-12.

The nucleotide sequence of the rpoD gene (encoding the primary sigma-70 (sigma 70) subunit of RNA polymerase) from Escherichia coli C was determined. This gene differs from that of E. coli K-12 by a 30-bp deletion and five single-bp substitutions, resulting in a sigma 70 subunit with three amino acid (aa) changes and a deletion of ten acidic aa.

Mutations affecting two adjacent amino acid residues in the alpha subunit of RNA polymerase block transcriptional activation by the bacteriophage P2 Ogr protein.

The bacteriophage P2 ogr gene product is a positive regulator of transcription from P2 late promoters. The ogr gene was originally defined by compensatory mutations that overcame the block to P2 growth imposed by a host mutation, rpoA109, in the gene encoding the alpha subunit of RNA polymerase. DNA sequence analysis has confirmed that this mutation affects the C-terminal region of the alpha subunit, changing a leucine residue at position 290 to a histidine (rpoAL290H). We have employed a reporter plasmid system to screen other, previously described, rpoA mutants for effects on activation of a P2 late promoter and have identified a second allele, rpoA155, that blocks P2 late transcription. This mutation lies just upstream of rpoAL290H, changing the leucine residue at position 289 to a phenylalanine (rpoAL289F). The effect of the rpoAL289F mutation is not suppressed by the rpoAL290H-compensatory P2 ogr mutation. P2 ogr mutants that overcome the block imposed by rpoAL289F were isolated and characterized. Our results are consistent with a direct interaction between Ogr and the alpha subunit of RNA polymerase and support a model in which transcription factor contact sites within the C terminus of alpha are discrete and tightly clustered.

Two genes for carbohydrate catabolism are divergently transcribed from a region of DNA containing the hexC locus in Pseudomonas aeruginosa PAO1.

The hexC locus of Pseudomonas aeruginosa PAO1 was localized to a 247-bp segment of chromosomal DNA on the multicopy broad-host-range vector pRO1614. The presence of this plasmid (pPZ196) in strain PAO1 produced the so-called "hexC effect," a two- to ninefold increase in the activities of four carbohydrate catabolism enzymes, glucokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase. The extent of the hexC effect was restricted, since three independently regulated metabolic enzymes were not affected by the presence of the hexC plasmid. Furthermore, the hexC-containing plasmid did not suppress catabolite repression control. Nucleotide sequence analysis of the segment of DNA encompassing hexC revealed a 128-bp region rich in adenosine-plus-thymine (AT) content separating two divergent open reading frames (ORFs). Transcriptional start sites for these two genes were mapped to the intergenic region, demonstrating that this sequence contained overlapping divergent promoters. The intergenic region contained potential regulatory sequences such as dyad symmetry motifs, polydeoxyadenosine tracts, and a sequence matching the integration host factor recognition site in Escherichia coli. One of the ORFs encoded a 610-amino-acid protein with 55 to 60% identity to 6-phosphogluconate dehydratase from E. coli and Zymomonas mobilis. The second ORF coded for a protein of 335 amino acids that displayed 45 to 60% identity to the NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAP) family of enzymes. The NAD-dependent GAP gene on the P. aeruginosa chromosome was previously unmapped. GAP was found to exhibit the hexC-dependent increase in its basal activity, establishing it as a fifth catabolic enzyme in the multioperonic hex regulon.

A merR homologue at 74 minutes on the Escherichia coli genome.

The nucleotide sequence between the trkA gene and the alpha ribosomal protein operon of Escherichia coli was determined. This 1592-bp region contains four open reading frames, one of which shows striking similarity to the MerR family of transcriptional regulatory proteins.

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.

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.

Mutational analysis of the bacteriophage P2 Ogr protein: truncation of the carboxy terminus.

The Ogr protein is a 72-residue, zinc-binding transcription factor essential for activation of late gene expression in bacteriophage P2. Analysis of C-terminal truncated proteins generated by stop codon mutagenesis shows that deletion of residues distal to position 51 had negligible effects on Ogr function. More-extensive deletion resulted in unstable products with severely reduced activity. These results, as well as the effects of other mutations in this region, support the idea that the 21 C-terminal residues are not required for transactivation.

Site-directed mutagenesis of an amino acid residue in the bacteriophage P2 ogr protein implicated in interaction with Escherichia coli RNA polymerase.

The P2 ogr gene encodes a 72-amino-acid protein required for P2 late gene expression. This gene was defined originally by a class of compensatory mutations which overcome the block to P2 late transcription imposed by a host mutation, rpoA109, in the gene encoding the alpha subunit of Escherichia coli RNA polymerase. Spontaneous compensatory ogr mutations substitute a Cys for a Tyr residue at amino acid 42 in the Ogr polypeptide. Using suppression of an ogr amber mutation and site-directed oligonucleotide mutagenesis, we have studied the effect of amino acid substitutions at this position in Ogr. Substitution of charged residues at this site renders Ogr protein inactive, in rpoA+ and rpoA109 strains. While 11 different amino acids are capable of replacing the wild-type Tyr-42 to allow P2 growth to varying degrees in a wild-type E. coli strain, only three of these allow phage growth in strains carrying the rpoA109 mutation. Phages carrying Cys or Ala in place of Tyr-42 gave burst sizes at least as high as P2 ogr+ in a rpoA+ strain; a Gly substitution also allowed P2 to grow in either a rpoA+ or rpoA109 background, but markedly reduced the burst size. These results are consistent with a direct interaction between Ogr and the alpha subunit of E. coli RNA polymerase in positive control of P2 late transcription, and indicate that the block imposed by the rpoA109 mutation is due to steric hindrance.

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.

Control of bacteriophage P2 gene expression: analysis of transcription of the ogr gene.

The bacteriophage P2 ogr gene encodes an 8.3-kDa protein that is a positive effector of P2 late gene transcription. The ogr gene is preceded by a promoter sequence (Pogr) resembling a normal Escherichia coli promoter and is located just downstream of a late transcription unit. We analyzed the kinetics and regulation of ogr gene transcription by using an ogr-specific antisense RNA probe in an S1 mapping assay. During a normal P2 infection, ogr gene transcription starts from Pogr at an intermediate time between the onset of early and late transcription. At late times after infection the ogr gene is cotranscribed with the late FETUD operon; the ogr gene product thus positively regulates its own synthesis from the P2 late promoter PF. Expression of the P2 late genes also requires P2 DNA replication. Complementation experiments and transcriptional analysis show that a nonreplicating P2 phage expresses the ogr gene from Pogr but is unable to transcribe the late genes. A P2 ogr-defective phage makes an increased level of ogr mRNA, consistent with autogenous control from Pogr. Transcription of the ogr gene in the prophage of a P2 heteroimmune lysogen is stimulated after infection with P2, suggesting that Pogr is under indirect immunity control and is activated by a yet-unidentified P2 early gene product during infection.

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.

Analysis of cloned structural and regulatory genes for carbohydrate utilization in Pseudomonas aeruginosa PAO.

Five of the genes required for phosphorylative catabolism of glucose in Pseudomonas aeruginosa were ordered on two different chromosomal fragments. Analysis of a previously isolated 6.0-kb EcoRI fragment containing three structural genes showed that the genes were present on a 4.6-kb fragment in the order glucose-binding protein (gltB)-glucokinase (glk)-6-phosphogluconate dehydratase (edd). Two genes, glucose-6-phosphate dehydrogenase (zwf) and 2-keto-3-deoxy-6-phosphogluconate aldolase (eda), shown by transductional analysis to be linked to gltB and edd, were cloned on a separate 11-kb BamHI chromosomal DNA fragment and then subcloned and ordered on a 7-kb fragment. The 6.0-kb EcoRI fragment had been shown to complement a regulatory mutation, hexR, which caused noninducibility of four glucose catabolic enzymes. In this study, hexR was mapped coincident with edd. A second regulatory function, hexC, was cloned within a 0.6-kb fragment contiguous to the edd gene but containing none of the structural genes. The phenotypic effect of the hexC locus, when present on a multicopy plasmid, was elevated expression of glucokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase activities in the absence of inducer.

Deletion analysis of a bacteriophage P2 late promoter.

We have fused the promoter (PF) for the P2 late FETUD operon to the gene (cat) encoding chloramphenicol acetyltransferase (CAT) in a plasmid vector. Synthesis of CAT in Escherichia coli strains carrying this plasmid requires the product of the P2 ogr gene or the satellite phage P4 transactivation gene, delta. Our results demonstrate that these phage-encoded transcriptional regulatory proteins are necessary and sufficient for activation of P2 late transcription in this reporter plasmid. Positive regulation of cloned PF is severely impaired in a host strain carrying the rpoA109 mutation. Expression from the cloned promoter thus approximates those features of P2 late transcription that have been shown to occur during normal P2 infection. To define sequences required for promoter function, sequential upstream deletions of PF were generated using BAL 31 nuclease, and the mutant promoters were assayed for cat expression. A sequence between nucleotides -69 and -64 from the transcription start point was found to be essential for promoter activity. This coincides with a region of homology conserved among all four P2 late gene promoters and the two P4 late promoters, and includes an element of dyad symmetry.

Purification and properties of the bacteriophage P2 ogr gene product. A prokaryotic zinc-binding transcriptional activator.

The bacteriophage P2 ogr gene product, a 72-residue basic protein rich in cysteine and histidine, is a positive regulatory factor for phage late gene transcription in both P2 and satellite phage P4. We have developed a simple purification procedure for Ogr protein synthesized from an overproducing plasmid. Inclusion bodies formed upon overproduction were denatured using 8 M urea, and the overproduced protein was purified by gel filtration. The purified Ogr was allowed to refold under optimized conditions and was subsequently shown to be able to transactivate the phage P4 late promoter Psid in an in vitro coupled transcription-translation system. Using a 65Zn blotting method and absorption spectroscopy, we show that Ogr is a zinc-binding protein and that the conserved cysteine residues are involved in forming a complex with Zn(II). The purification procedure described allows Ogr to be obtained in both high purity and yield.