Gene insertion and replacement in Schizosaccharomyces pombe mediated by the Streptomyces bacteriophage phiC31 site-specific recombination system.

The site-specific recombination system used by the Streptomyces bacteriophage phiC31 was tested in the fission yeast Schizosaccharomyces pombe. A target strain with the phage attachment site attP inserted at the leu1 locus was co-transformed with one plasmid containing the bacterial attachment site attB linked to a ura4+ marker, and a second plasmid expressing the phiC31 integrase gene. High-efficiency transformation to the Ura+ phenotype occurred when the integrase gene was expressed. Southern analysis revealed that the attB-ura4+ plasmid integrated into the chromosomal attP site. Sequence analysis showed that the attBxattP recombination was precise. In another approach, DNA with a ura4+ marker flanked by two attB sites in direct orientation was used to transform S. pombe cells bearing an attP duplication. The phiC31 integrase catalyzed two reciprocal cross-overs, resulting in a precise gene replacement. The site-specific insertions are stable, as no excision (the reverse reaction) was observed on maintenance of the integrase gene in the integrant lines. The irreversibility of the phiC31 site-specific recombination system sets it apart from other systems currently used in eukaryotic cells, which reverse readily. Deployment of the phiC31 recombination provides new opportunities for directing transgene and chromosome rearrangements in eukaryotic systems.

Evidence for an active role of the DnaK chaperone system in the degradation of sigma(32).

Under non-stressed conditions in Escherichia coli, the heat shock transcription factor sigma(32) is rapidly degraded by the AAA protease FtsH. The DnaK chaperone system is also required for the rapid turnover of sigma(32) in the cell. It has been hypothesized that the DnaK chaperone system facilitates the degradation of sigma(32) by sequestering it from RNA polymerase core. This hypothesis predicts that mutant sigma(32) proteins, which are deficient in binding to RNA polymerase core, will be degraded independently of the DnaK chaperone system. We examined the in vivo stability of such mutant sigma(32) proteins. Results indicated that the mutant sigma(32) proteins as similar as authentic sigma(32) were stabilized in DeltadnaK and DeltadnaJ/DeltacbpA cells. The interaction between sigma(32) and DnaK/DnaJ/GrpE was not affected by these mutations. These results strongly suggest that the degradation of sigma(32) requires an unidentified active role of the DnaK chaperone system.

Complete nucleotide sequence, molecular analysis and genome structure of bacteriophage A118 of Listeria monocytogenes: implications for phage evolution.

A118 is a temperate phage isolated from Listeria monocytogenes. In this study, we report the entire nucleotide sequence and structural analysis of its 40 834 bp DNA. Electron microscopic and enzymatic analyses revealed that the A118 genome is a linear, circularly permuted, terminally redundant collection of double-stranded DNA molecules. No evidence for cohesive ends or for a terminase recognition (pac) site could be obtained, suggesting that A118 viral DNA is packaged via a headful mechanism. Partial denaturation mapping of DNA cross-linked to the tail shaft indicated that DNA packaging proceeds from left to right with respect to the arbitrary genomic map and the direction of genes necessary for lytic development. Seventy-two open reading frames (ORFs) were identified on the A118 genome, which are apparently organized in a life cycle-specific manner into at least three major transcriptional units. N-terminal amino acid sequencing, bioinformatic analyses and functional characterizations enabled the assignment of possible functions to 26 ORFs, which included DNA packaging proteins, morphopoetic proteins, lysis components, lysogeny control-associated functions and proteins necessary for DNA recombination, modification and replication. Comparative analysis of the A118 genome structure with other bacteriophages revealed local, but sometimes extensive, similarities to a number of phages spanning a broader phylogenetic range of various low G+C host bacteria, which implies relatively recent exchange of genes or genetic modules. We have also identified the A118 attachment site attP and the corresponding attB in Listeria monocytogenes, and show that site-specific integration of the A118 prophage by the A118 integrase occurs into a host gene homologous to comK of Bacillus subtilis, an autoregulatory gene specifying the major competence transcription factor.

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.

A complex control system for transcriptional activation from the sid promoter of bacteriophage P4.

The sid gene promoter (Psid), which controls expression of the late genes from satellite phage P4, is activated by a unique class of small DNA-binding proteins. The activators from both satellite and helper phages stimulate transcription from Psid. These activators bind to sites centered at position -55 in all the helper and satellite phage late promoters. P4 Psid is unique in that it has an additional activator binding site centered at position -18 (site II). We have constructed a mutant of site II that no longer binds activators. Transcription under the control of satellite phage activators is increased by the site II mutation. In contrast, helper phage activators do not show this increase in transcription from Psid mutated at site II. Competition gel shift analysis reveals that the P4 satellite phage activator, Delta, binds eightfold better to site II than to site I. The products of the sid transcription unit are needed only when a helper phage is present; thus, the satellite phage activators repress transcription until the helper is present to supply a nonrepressing activator.

Multiple regions on the Escherichia coli heat shock transcription factor sigma32 determine core RNA polymerase binding specificity.

We have analyzed the core RNA polymerase (RNAP) binding activity of the purified products of nine defective alleles of the rpoH gene, which encodes sigma32 in Escherichia coli. All mutations studied here lie outside of the putative core RNAP binding regions 2.1 and 2.2. Based on the estimated K(s)s for the mutant sigma and core RNAP interaction determined by in vitro transcription and by glycerol gradient sedimentation, we have divided the mutants into three classes. The class III mutants showed greatly decreased affinity for core RNAP, whereas the class II mutants' effect on core RNAP interaction was only clearly seen in the presence of sigma70 competitor. The class I mutant behaved nearly identically to the wild type in core RNAP binding. Two point mutations in class III altered residues that were distant from one another. One was found in conserved region 4.2, and the other was in a region conserved only among heat shock sigma factors. These data suggest that there is more than one core RNAP binding region in sigma32 and that differences in contact sites probably exist among sigma factors.

The helicase domain of phage P4 alpha protein overlaps the specific DNA binding domain.

Replication initiation depends on origin recognition, helicase, and primase activities. In phage P4, a second DNA region, the cis replication region (crr), is also required for replication initiation. The multifunctional alpha protein of phage P4, which is essential for DNA replication, combines the three aforementioned activities on a single polypeptide chain. Protein domains responsible for the activities were identified by mutagenesis. We show that mutations of residues G506 and K507 are defective in vivo in phage propagation and in unwinding of a forked helicase substrate. This finding indicates that the proposed P loop is essential for helicase activity. Truncations of gene product alpha (gp alpha) demonstrated that 142 residues of the C terminus are sufficient for specifically binding ori and crr DNA. The minimal binding domain retains gp alpha's ability to induce loop formation between ori and crr. In vitro and in vivo analysis of short C-terminal truncations indicate that the C terminus is needed for helicase activity as well as for specific DNA binding.

Cnr protein, the negative regulator of bacteriophage P4 replication, stimulates specific DNA binding of its initiator protein alpha.

Bacteriophage P4 DNA replication depends upon the phage-encoded alpha protein, which has DNA helicase and DNA primase activity and can specifically bind to the replication origin (ori) and to the cis replicating region (crr). The P4 Cnr protein functions as a negative regulator of P4 replication, and P4 does not replicate in cells that overexpress cnr. We searched for P4 mutants that suppressed this phenotype (Cnr resistant [alpha cr]). Eight independent mutants that grew in the presence of high levels of Cnr were obtained. None of these can establish the plasmid state. Each of these mutations lies in the DNA binding domain of gp alpha that occupies the C terminus of the protein. Five different sequence changes were found: T675M, G732V (three times), G732W (twice), L733V, and L737V. A TrxA-Cnr fusion protein does not bind DNA by itself but stimulates the ori and crr binding abilities of alpha protein in vitro. The alpha cr mutant proteins were still able to bind specifically to ori or crr, but specific DNA binding was less stimulated by the TrxA-Cnr protein. We present evidence that Cnr protein interacts with the gp alpha domain that binds specifically to DNA and that gp(alpha)cr mutations impair this interaction. We hypothesize that gp alpha-Cnr interaction is essential for the control of P4 DNA replication.

A sigma32 mutant with a single amino acid change in the highly conserved region 2.2 exhibits reduced core RNA polymerase affinity.

sigma32, the product of the rpoH gene in Escherichia coli, provides promoter specificity by interacting with core RNAP. Amino acid sequence alignment of sigma32 with other sigma factors in the sigma70 family has revealed regions of sequence homology. We have investigated the function of the most highly conserved region, 2.2, using purified products of various rpoH alleles. Core RNAP binding analysis by glycerol gradient sedimentation has revealed reduced core RNAP affinity for one of the mutant sigma32 proteins, Q80R. This reduced core interaction is exacerbated in the presence of sigma70, which competes with sigma32 for binding of core RNAP. When a different but more conserved amino acid was introduced at this position by site-directed mutagenesis (Q80N), this mutant sigma factor still displayed a significant reduction in its core RNAP affinity. Based on these results, we conclude that at least one specific amino acid in region 2.2 is involved in core RNAP interaction.

The two P2 Ogr-like domains of the delta protein from bacteriophage P4 are required for activity.

The satellite P4 phage Delta protein positively regulates the late genes of its helper bacteriophage P2, as well as its own late genes. Delta is a member of a class of activators associated with P2-or P4-like phages and is the largest member of this family. It resembles a covalently joined head-to-tail dimer of the other members of this family of activators. We have analyzed the requirement for both standard domains of Delta through the isolation of amber mutants and the insertion of amber linkers. We show that both domains of Delta are required for DNA binding in vivo and for transcriptional activity. Proper spacing between the two domains is important for activity at two of the four P2 promoters. Expression of both domains from different plasmids causes activation of late gene transcription in vivo of all six late promoters of P2 and P4. A monomric Delta from another satellite phage, phi R73, can function efficiently as a covalent dimer but when this Delta is made dimeric with the second half of P4 delta, it activates less efficiently.

A novel mechanism of virus-virus interactions: bacteriophage P2 Tin protein inhibits phage T4 DNA synthesis by poisoning the T4 single-stranded DNA binding protein, gp32.

P2 prophages have been known to inhibit DNA replication and growth of T-even phages. We show here that this inhibition is due to poisoning of the T-even single-stranded DNA binding protein gp32 by the product of the nonessential P2 tin gene. Synthesis of Tin protein from a gene cloned in a multicopy plasmid is necessary and sufficient to completely prevent de novo DNA replication and growth of wild-type T2 or T4 phage. We isolated more than 20 independent mutants that render T-even phages resistant to poisoning by the P2 Tin protein. In all of these mutants, which we call asp, Asp codon 163 of gene 32 is changed to a Gly or Asn codon. The mutant alleles are recessive; i.e., when wild-type and asp mutants coinfect the same host cells, most DNA replication is poisoned by P2 Tin protein. To explain our results, we propose that the P2 Tin protein interacts with T-even gp32 at position 163 and distorts the helical filament of gene 32 protein on single-stranded DNA. Thereby Tin protein inhibits either assembly or function, or both, of the T4 replisome. The inhibition of late gene expression by P2 Tin protein may be an indirect consequence of inhibition of DNA replication.

In vivo and in vitro evidence for an anti-Rho activity induced by the phage P4 polarity suppressor protein Psu.

The polarity suppression (Psu) protein of bacteriophage P4 causes suppression of transcriptional polarity in Escherichia coli by overcoming Rho termination factor activity. Two new psu mutants defective in polarity suppression are described. The psu5 mutation deletes codons 95-98 from about the middle of the gene, and the mutant protein is inactive. The psu6 mutation changes Phe169 to Val and encodes a temperature-sensitive protein. Constitutive overexpression of psu+ from a plasmid prevents colony formation, but overexpression of mutant genes (psu5, psu6) does not, suggesting that Psu disturbs essential host function(s). Rho protein synthesis is enhanced several-fold in cells containing wild-type Psu, due to readthrough at the rho attenuator, while the physical stability of Rho is maintained. As a consequence, Psu-producing cells accumulate significantly more Rho than normal cells, reminiscent of termination-defective rho mutants. The polarity suppression activity induced by Psu is demonstrated in vitro by the efficient readthrough of Rho-dependent terminators lambda tR1 and TIS2 during coupled transcription-translation. Purified Rho protein restores termination at TIS2 when added to Psu-containing reactions but NusG does not. The data support the hypothesis that Psu has or elicits an anti-Rho function.

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.

Bacteriophage PSP3 and phiR73 activator proteins: analysis of promoter specificities.

Transcription from the late promoters of bacteriophage P2 and its satellite phage P4 is activated by a unique class of small, zinc-binding proteins. Using plasmid expression systems, we compared activators from two P2-like (helper) phages with those encoded by two satellite phages. The helper phage activators have more activity on the P4 phage sid promoter. In contrast, the satellite phage activators function better on the four late P2 promoters and on the P4 late leftward promoter. We purified one activator encoded by a P2-like phage and an activator from a satellite phage and determined their binding sites within the P2 and P4 late promoters. Differences in activity levels correlate with binding specificities; promoters that function best with the satellite phage activators have only one activator binding site centered at -55, while the P4 sid promoter, which has more activity with helper phage activators, has a second binding site centered at -18. Surprisingly, DNase I footprinting revealed only very minor differences in promoter binding by the two activators reported here and the P4 activator reported previously. Thus, the differences in transcriptional activity are probably due to interactions between the activators and RNA polymerase, rather than interactions between the activators and DNA.

Domain structure of phage P4 alpha protein deduced by mutational analysis.

Bacteriophage P4 DNA replication depends on the product of the alpha gene, which has origin recognition ability, DNA helicase activity, and DNA primase activity. One temperature-sensitive and four amber mutations that eliminate DNA replication in vivo were sequenced and located in the alpha gene. Sequence analysis of the entire gene predicted a domain structure for the alpha polypeptide chain (777 amino acid residues, M(r) 84,900), with the N terminus providing the catalytic activity for the primase and the middle part providing that for the helicase/nucleoside triphosphatase. This model was confirmed experimentally in vivo and in vitro. In addition, the ori DNA recognition ability was found to be associated with the C-terminal third of the alpha polypeptide chain. The type A nucleotide-binding site is required for P4 replication in vivo, as shown for alpha mutations at G-506 and K-507. In the absence of an active DnaG protein, the primase function is also essential for P4 replication. Primase-null and helicase-null mutants retain the two remaining activities functionally in vitro and in vivo. The latter was demonstrated by trans complementation studies, indicating the assembly of active P4 replisomes by a primase-null and a helicase-null mutant.

Purification and characterization of the bacteriophage P4 delta protein.

The bacteriophage P4 delta protein is a transcriptional activator of the late genes of P4 as well as the late genes of its helpers, such as bacteriophage P2. delta was purified, using a variation of the MalE fusion system. With this method we purified two forms of delta: a fusion of MalE and delta and a unfused form. The fusion by itself is not active in vivo or in vitro, but the mixture of the fusion and the unfused delta is active in both. Using nitrocellulose filtration and gel mobility shift assays, we show that delta binds DNA, and using DNase I footprinting, we show that delta binds to sequences centered at approximately -55 in the two late promoters of P4 as well as the four late promoters of its helper P2. In addition, the P4 sid promoter contains a second delta binding site centered at -18.

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.