Characterization of Plys-proximal morphogenetic genes of transposable bacteriophage Mu.

Late during the bacteriophage Mu lytic cycle, Mu DNA must be matured and packaged from its dispersed integration sites in the host DNA in order to produce progeny virions. Whereas control of late gene transcription in Mu is becoming well understood, less is known about the phage morphogenetic process. To investigate the latter, we cloned and sequenced a approximately 4.3-kb region of the phage DNA beginning just upstream of the leftmost late promoter Plys. Previous mapping of amber mutations had located the lysis (lys) and proposed DNA maturation genes D and E in this region. When the DNA sequence was analyzed, seven potential open reading frames were found. DNA sequence analysis of amber mutations in genes D and E identified the sixth and seventh open reading frames as D and E, respectively. Cloning and expression of this region enabled production of cell-free protein extracts that specifically recognize the phage-encoded packaging sequence (pac), a characteristic exhibited by phage maturation enzymes. In addition, the E protein was found to share homology with the large subunit of many phage DNA maturation enzymes. These results support the hypothesis that D and E encode subunits of the Mu DNA maturation enzyme.

Characterization of in vitro DNA binding sites of the EUO protein of Chlamydia psittaci.

The EUO gene of chlamydia is highly expressed early in the developmental cycle, relative to other genes, but continues to be expressed throughout the active growth phases. The precise function of EUO protein is not known, but it binds to DNA in vitro. In this study, we developed a selection and amplification scheme for identifying chlamydial genomic fragments to which EUO preferentially binds in vitro. The scheme involved mixing recombinant EUO with a Chlamydia psittaci genomic library in a pBluescript plasmid vector in vitro, trapping EUO-bound plasmid clones on filters, and amplifying the clones in Escherichia coli. After nine rounds of enrichment, the EUO binding sites of the three most highly enriched clones were identified by DNase I footprint analysis. All three clones had multiple binding sites of various sizes with no clear distinguishing feature other than they were AT-rich and were usually not located in putative promoter regions. We used limited site-specific mutagenesis to characterize the strongest binding site of the most-highly-enriched clone, which represented about 50% of the population after nine rounds. This mutagenesis identified a core binding site of 15 nucleotides (nt) whose sequence was used to find related sequences within each of the strong binding sites in the other two clones. Using the frequency of bases at specific positions within this group of sequences as a guide, we carried out trial-and-error searching with many related sequences, eliminating those which identified nonfootprinted sites. This process led us to the consensus 15-nt sequence AHGAAAWVTYTWDAY, which, when allowing two mismatches, picked out all of the strong binding sites and no nonfootprinting sites within the three enriched clones. This sequence may be useful for predicting additional possible EUO binding sites in the chlamydial genome.

Discovery of the tail tube gene of bacteriophage Mu and sequence analysis of the sheath and tube genes.

The nucleotide sequence was determined for 2.75 kbp of phage Mu DNA encoding the contractile tail sheath protein L. N-terminal sequence analysis of Mu tail tube and sheath proteins identified the open reading frame just downstream of gene L as the tube gene. This clustering and order of the sheath and tube genes appear to be common among the myoviridae. Database homology searches revealed high similarity between the Mu sheath and tube proteins and two proteins in a Haemophilus influenzae Mu-like prophage, suggesting that they are the sheath and tube proteins of that prophage.

Transcription activation by the bacteriophage Mu Mor protein requires the C-terminal regions of both alpha and sigma70 subunits of Escherichia coli RNA polymerase.

Middle transcription of bacteriophage Mu requires Escherichia coli RNA polymerase and a Mu-encoded protein, Mor. Consistent with these requirements, the middle promoter, Pm, has a -10 hexamer but lacks a recognizable -35 hexamer. Interactions between Mor and RNA polymerase were studied using in vitro transcription, DNase I footprinting, and the yeast interaction trap system. We observed reduced promoter activity in vitro using reconstituted RNA polymerases with C-terminal deletions in alpha or sigma70. As predicted if alpha were binding to Pm, we detected a polymerase-dependent footprint in the -60 region. Reconstituted RNA polymerases containing Ala substitutions in the alpha C-terminal domain were used to assay Mor-dependent transcription from Pm in vitro. The D258A substitution and alpha deletion gave large reductions in activation, whereas the L262A, R265A, and N268A substitutions caused smaller reductions. The interaction trap assay revealed weak interactions between Mor and both alpha and sigma70; consistent with a key role of alpha-D258, the D258A substitution abolished interaction, whereas the R265A substitution did not. We propose that: (i) alpha-D258 is a Mor "contact site"; and (ii) residues Leu-262, Arg-265, and Asn-268 indirectly affect Mor-polymerase interaction by stabilizing the ternary complex via alpha-DNA contact.

Distortion in the spacer region of Pm during activation of middle transcription of phage Mu.

Transcription from the middle promoter, Pm, of phage Mu is initiated by Escherichia coli RNA polymerase holoenzyme (E sigma 70; RNAP) and the phage-encoded activator, Mor. Point mutations in the spacer region between the -10 hexamer and the Mor binding site result in changes of promoter activity in vivo. These mutations are located at the junction between a rigid T-tract and adjacent, potentially deformable G + C-rich DNA segment, suggesting that deformation of the spacer region may play a role in the transcriptional activation of Pm. This prediction was tested by using dimethyl sulfate and potassium permanganate footprinting analyses. Helical distortion involving strand separation was detected at positions -32 to -34, close to the predicted interface between Mor and RNAP. Promoter mutants in which this distortion was not detected exhibited a lack of melting in the -12 to -1 region and reduced promoter activity in vivo. We propose that complexes containing the distortion represent stressed intermediates rather than stable open complexes and thus can be envisaged as a transition state in the kinetic pathway of Pm activation in which stored torsional energy could be used to facilitate melting around the transcription start point.

Regulatory factors acting at the bacteriophage Mu middle promoter.

Lytic development of bacteriophage Mu proceeds through three phases of transcription: early, middle, and late. Initiation of middle transcription from Pm requires the phage-encoded activator, Mor. An examination of the sequences surrounding the promoter revealed possible binding sites for Mu proteins A and c, as well as for Escherichia coli integration host factor. Promoter fragments containing 5' and 3' deletions were fused to the lacZ reporter gene and assayed for activity after induction of a Mu prophage or a plasmid-borne mor gene. Sequences upstream of position -62 and downstream of +10 were dispensable for promoter activity. In DNase I footprinting with both crude extract and purified protein, Mor protected Pm sequences from position -56 to -33. Mutations disrupting the dyad symmetry of the terminator of early transcription overlapping the Mor binding site did not reduce promoter activity, suggesting that the symmetry per se is not required for Mor binding or Pm activation. Purified Mu lysogenic repressor (c) also bound to Pm, overlapping the Mor binding site. Production of large amounts of repressor in vivo reduced Mor-dependent promoter activity nearly 10-fold. Promoters with mutations in the repressor binding site showed a reduction in this repressor-mediated inhibition of Pm activity.

Transcription activation by the bacteriophage Mu Mor protein: analysis of promoter mutations in Pm identifies a new region required for promoter function.

Middle transcription of bacteriophage Mu requires Escherichia coli RNA polymerase holoenzyme and a Mu-encoded protein, Mor. Consistent with these requirements, the middle promoter, Pm, has a recognizable -10 region but lacks a -35 region. Mutagenesis of this promoter (from -70 to +10) was performed using mutagenic oligonucleotide-directed PCR. The resulting fragments were cloned into a promoter-lacZfusion vector and analyzed for promoter activity by assaying beta-galactosidase production. Single point mutations with a Down phenotype were clustered in three regions: the -10 region, the Mor footprint region and the spacer between them. Gel retardation experiments with purified Mor protein and promoter mutants demonstrated that sequences important for Mor binding are located within the Mor footprint region and lead us to propose the existence of a dyad symmetry element involved in Mor binding. In agreement with this prediction, glutaraldehyde crosslinking of Mor in solution generated a species with the size of a dimer. These experiments also identified an unusual group of mutations located in the spacer region adjacent to the Mor footprint. These mutations alter promoter activity without affecting Mor binding. A circular permutation assay revealed that Mor does not introduce a significant bend upon binding to its target sequence.

Cloning, sequence and in vitro transcription/translation analysis of a 3.2-kb EcoRI-HindIII fragment of Leuconostoc oenos bacteriophage L10.

A 3.2-kb EcoRI-HindIII DNA fragment of Leuconostoc oenos bacteriophage L10 was cloned and sequenced. Computer-assisted analysis of the sequence identified eleven possible open reading frames (ORFs) that were all on the same strand. In vitro transcription/translation analysis of the full-length DNA fragment yielded five prominent proteins that were correlated with ORFs by their sizes and expression from deleted clones. Only those ORFs containing recognizable Shine-Dalgarno sequences coded for proteins. Neither the nucleotide sequence, nor deduced amino-acid sequences showed significant homology with other known sequences.

Identification and characterization of the terminators of the lys and P transcripts of bacteriophage Mu.

Transcription during the lytic cycle of phage Mu occurs in three phases: early, middle, and late. Late transcription requires the Mu C protein and initiates at four promoters: Plys, PI, PP, and Pmom. Northern blot analysis of total RNA isolated 30 min after heat induction of Mu cts lysogens demonstrated that the full-length lys and P transcripts were approximately 7.6 and 6.3 kb long, respectively. The 3' ends of the lys and P transcripts were further localized by S1 nuclease mapping to intergenic regions between G and I and between U and U' in both the G(+) and G(-) orientations of the invertible G segment, respectively. As expected, when DNA fragments containing these termination regions were cloned into plasmids between Pgal and the galK gene, they showed efficient termination activity, even in a Rho-deficient background. Deletion analysis indicated that efficient termination required the presence of potential RNA stem-loop structures immediately preceding the RNA 3' ends. For the P transcript from phage with the G(-) orientation, full termination activity required both the region containing the stem-loop structure and upstream sequences. Taken together, these results suggest that the transcription termination sites of the lys and P transcripts are Rho-independent terminators.

Mutational analysis of a C-dependent late promoter of bacteriophage Mu.

Late transcription of bacteriophage Mu initiates at four promoters, P(lys), PI, PP and Pmom, and requires the Mu C protein and the host RNA polymerase. Promoter-containing DNA fragments extending approximately 200 bp upstream and downstream of the 5' starts of the lys, I and P transcripts were cloned into a multicopy lacZ-expression plasmid. Promoter activity, assayed by beta-galactosidase expression, was determined under two different conditions: (1) with C provided from a compatible plasmid in the absence of other Mu factors and (2) with C provided from an induced Mu prophage. beta-galactosidase activities were greatest for P(lys), intermediate for PI, and lowest for PP. Similar analysis of plasmids containing nested sets of deletions removing 5' or 3' sequences of P(lys) demonstrated that a 68-bp region was sufficient for full activity. Point mutations were generated within the 68-bp region by mutagenic oligonucleotide-directed PCR (Mod-PCR). Properties of the lys promoter mutants indicated that, in addition to the -10 region, a 19-bp region from -52 to -34 containing the C footprint is required for C-dependent promoter activity.

The bacteriophage Mu middle operon: essential and nonessential functions.

Transcription during the bacteriophage Mu lytic cycle occurs in three phases: early, middle, and late. Previous DNA sequence analysis of the middle operon revealed five potential open reading frames (ORFs) with lengths of 39, 42, 72, 120, and 140 amino acids. The distal 140-amino-acid ORF encodes C, the activator of late transcription. Expression of the middle operon under the control of a T7 promoter and T7 RNA polymerase resulted in production of two polypeptides of 15 (ORF 120) and 16.5 kDa (C). Introduction of a linker containing a translation terminator into ORF120 resulted in the production of a truncated form of the ORF120 polypeptide. When the ORF120 linker mutation and several middle operon deletion mutations were assayed for their effect on Mu growth in Escherichia coli K12, the deletions caused 6- to 22-min delays in lysis, and two resulted in a smaller plaque morphology, but all gave normal plating efficiencies and burst sizes. The plating efficiencies for all the mutants were also similar to that of wild-type Mu on alternate hosts E. coli C, Citrobacter freundii, Shigella sonnei, and Shigella flexneri. These results indicate that, with the exception of C, the middle operon ORFs are not essential for phage development.

Bacteriophage Mu Mor protein requires sigma 70 to activate the Mu middle promoter.

Transcription during the bacteriophage Mu lytic cycle occurs in three phases: early, middle, and late. Middle transcription requires the early gene product Mor for its activation. Mor protein overproduction was accomplished by fusing the mor gene to an efficient phage T7 promoter and translation initiation region. A protein fraction highly enriched for Escherichia coli RNA polymerase (E sigma 70) from the Mor-overproducing strain was able to activate transcription from both the tac promoter (Ptac) and the Mu middle promoter (Pm) in vitro. Transcription initiation from Pm was Mor dependent, and the RNA 5' end was identical to that of in vivo RNA. Addition of anti-sigma 70 antibody to transcription reactions containing Ptac and Pm resulted in inhibition of transcription from both promoters; addition of purified sigma 70 restored transcription. These results indicate that Mor-dependent activation requires sigma 70 and therefore imply that Mor is not an alternate sigma factor. This conclusion was further substantiated by a reconstitution experiment with purified proteins in which all three components, Mor, sigma 70, and core RNA polymerase, were required for Pm-dependent transcription in vitro. The sigma 70 dependence of Mor-specific transcription and the amino acid sequence similarity between Mor and C (an activator for Mu late transcription) both support the hypothesis that Mor functions mechanistically as an activator protein.

Mutagenic oligonucleotide-directed PCR amplification (Mod-PCR): an efficient method for generating random base substitution mutations in a DNA sequence element.

Saturation mutagenesis is one approach for determining the contributions of individual base pairs to the structure and function of defined DNA sequence elements. In this paper, we describe a novel method for saturation mutagenesis involving PCR amplification with degenerate synthetic oligonucleotides as primers. The degeneracy is confined to a specific target within the primer by mixing a low percentage of the three non-wild type (non-WT) nucleotide precursors with WT at specific positions during primer synthesis. PCR amplification of WT template DNA with the degenerate primer and an opposing WT primer, followed by subsequent cloning using restriction sites designed into the primers, results in recovery of a population of randomly mutated products. Since primers with multiple mutations hybridize less efficiently to WT template DNA during PCR amplification, the recovery of mutants with multiple base changes is greatly reduced. The efficient generation of random point mutations with this method allows the construction of separate mutant populations, each mutagenized over a different portion of the DNA sequence element. If a phenotypic assay is available, these populations can be screened directly to define those regions within the element that are important for activity. Only those populations containing mutations in the important regions require further characterization by DNA sequence analysis.

DNA sequence characterization of the G gene region of bacteriophage Mu.

The nucleotide sequence of a 1.2 kb region of bacteriophage Mu DNA was determined. This region contains the 3' end of the F gene, the complete G gene, and the 5' end of the I gene, all late genes involved in Mu virion morphogenesis. Identity of the G gene open reading frame was confirmed by sequencing four Gam mutations. The G open reading frame is predicted to encode proteins of 16.7 or 17.2 kDa, depending on which of two possible start codons are used to initiate translation. Four new nuB mutations in the DNA gyrase-binding site between the G and I genes were also sequenced and found to be identical to the nuB103 mutation sequenced previously.

Temperature-sensitive mutations in the bacteriophage Mu c repressor locate a 63-amino-acid DNA-binding domain.

Phage Mu's c gene product is a cooperative regulatory protein that binds to a large, complex, tripartite 184-bp operator. To probe the mechanism of repressor action, we isolated and characterized 13 phage mutants that cause Mu to undergo lytic development when cells are shifted from 30 to 42 degrees C. This collection contained only four mutations in the repressor gene, and all were clustered near the N terminus. The cts62 substitution of R47----Q caused weakened specific DNA recognition and altered cooperativity in vitro. A functional repressor with only 63 amino acids of Mu repressor fused to a C-terminal fragment of beta-galactosidase was constructed. This chimeric protein was an efficient repressor, as it bound specifically to Mu operator DNA in vitro and its expression conferred Mu immunity in vivo. A DNA looping model is proposed to explain regulation of the tripartite operator site and the highly cooperative nature of repressor binding.

Identification of a positive regulator of the Mu middle operon.

Transcription of bacteriophage Mu occurs in a regulatory cascade consisting of three phases: early, middle, and late. The 1.2-kb middle transcript is initiated at Pm and encodes the C protein, the activator of late transcription. A plasmid containing a Pm-lacZ operon fusion was constructed. beta-Galactosidase expression from the plasmid increased 23-fold after Mu prophage induction. Infection of plasmid-containing cells with lambda phages carrying different segment of the Mu early region localized the Pm-lacZ transactivation function to the region containing open reading frames E16 and E17. Deletion and linker insertion analyses of plasmids containing this region identified E17 as the transactivator; therefore we call this gene mor, for middle operon regulator. Expression of mor under the control of a T7 promoter and T7 RNA polymerase resulted in the production of a single polypeptide of 17 kDa as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Insertion of a linker into mor substantially reduced the ability of Mu to form plaques. When growth of the mor mutant was assayed in liquid, lysis was delayed by about 50 min and the burst size was approximately one-fifth that of wild-type Mu. The mor requirement for plaque formation and normal growth kinetics was abolished when C protein was provided in trans, indicating that the primary function of Mor is to provide sufficient C for late gene expression. Comparison of the predicted amino acid sequence of Mor with other proteins revealed that Mor and C share substantial amino acid sequence homology.

A DNA gyrase-binding site at the center of the bacteriophage Mu genome is required for efficient replicative transposition.

We have discovered a centrally located site that is required for efficient replication of bacteriophage Mu DNA and identified it as a strong DNA gyrase-binding site. Incubation of Mu DNA with gyrase and enoxacin revealed a cleavage site 18.1 kilobases from the left end of the 37.2-kilobase genome. Two observations indicate a role for the site in Mu replication: mutants of Mu, able to grow on an Escherichia coli gyrB host that does not allow growth of wild-type Mu, were found to possess single-base changes resulting in more efficient gyrase binding and cleavage at the site. Introduction of a 147-base-pair deletion that eliminated the site from a prophage inhibited the onset of Mu replication for greater than 1 hr after induction.

Bacteriophage Mu sites and functions involved in the inhibition of lambda::mini-Mu growth.

To better understand the nature of the mini-Mu-directed process which results in inhibition of lambda::mini-Mu growth we characterized spontaneous deletion mutants of the lambda::mini-Mu phage. On the basis of analysis of the deletion endpoints, mini-Mu replication functions, and integration and inhibition properties, the lambda::mini-Mu deletion mutants were divided into five classes which define the Mu sites and functions involved in lambda::mini-Mu growth inhibition. Class 1 mutants, which still exhibit lambda::mini-Mu growth inhibition, collectively delete all the Mu late functions encoded by the mini-Mu. Class 2 and 5 mutants, which show cis-dominant defects in inhibition and integration, delete the right and left mini-Mu attachment sites, respectively. Phages of Classes 3 and 4, which delete the Mu B or A and B genes, respectively, show recessive defects in growth inhibition. The properties of these mutants define the Mu replication functions, A and B, and the Mu attachment sites as essential for the inhibition of lambda::mini-Mu growth. The observation that the sites and functions essential for Mu replication also have requisite roles in the inhibition of lambda::mini-Mu growth suggests that inhibition results from mini-Mu-promoted replicative interference of lambda::mini-Mu development.

Activation of the bacteriophage Mu lys promoter by Mu C protein requires the sigma 70 subunit of Escherichia coli RNA polymerase.

Bacteriophage Mu C protein, a product of the middle operon, is required for activation of the four Mu late promoters. To address its mechanism of action, we overproduced the approximately 16.5-kilodalton C protein from a plasmid containing the C gene under the control of a phage T7 promoter and ribosome-binding site. A protein fraction highly enriched for Escherichia coli RNA polymerase (E sigma 70) and made from the overproducing strain was able to activate transcription in vitro from both the tac promoter (Ptac) and a Mu late promoter, Plys. The behavior of Plys was similar in vivo and in vitro; under both conditions, transcription was C dependent and the RNA 5' ends were identical. When anti-sigma 70 antibody was added to C-dependent transcription reactions containing both Ptac and Plys templates, transcription from both promoters was inhibited; transcription was restored by the addition of excess E sigma 70. This result suggests that C-dependent activation of Plys requires sigma 70. Further supporting evidence was provided by a reconstitution experiment in which an E sigma 70-depleted fraction containing C was unable to activate transcription from Plys unless both purified sigma 70 and core polymerase were added. These results strongly suggest that C is not a new sigma factor but acts as an activator for E sigma 70-dependent transcription.

Characterization of the C operon transcript of bacteriophage Mu.

Mu transcription occurs in three phases: early, middle, and late. Middle transcription occurs in the region of the C gene, which encodes the transactivator for late transcription. A middle promoter, Pm, was previously localized between 0.28 and 1.2 kilobase pairs upstream of C. We used S1 nuclease mapping with both unlabeled and radiolabeled capped RNAs from induced lysogens to characterize C transcription and identify its promoter. The C transcription initiation site was localized to a 4-base-pair region, approximately 740 base pairs upstream of C within the region containing Pm. Transcription of C was activated between 4 and 8 min after induction of cts and Cam lysogens and increased throughout the lytic cycle. Significant C transcription did not occur in replication-defective Aam lysogens. These kinetic and regulatory characteristics identify the C transcript as a middle RNA species and demonstrate that Pm is the C promoter. DNA sequence analysis of the Pm region showed a good -10, but poor -35, site homology to the Escherichia coli RNA polymerase consensus sequence. In addition, the sequence demonstrated that C is the distal gene in a middle operon containing several open reading frames. S1 mapping also showed an upstream transcript with a 3' end in the Pm region at a sequence strongly resembling a Rho-independent terminator. The regulatory characteristics of this RNA are consistent with this terminator, t9.2, being the early operon terminator.