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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.

Bacteriophage D3112 is a Mu-like temperate transposable phage of Pseudomonas aeruginosa. Genetic mapping and DNA sequence analysis have identified the left end of the phage genome as encoding the transposase enzyme (A) and the lysogenic (c) repressor. The c open reading frame (ORF), located at the leftmost end of the phage genome and transcribed from right to left, has four possible GTG initiation codons. Using site-directed mutagenesis, each of the four GTG codons was modified to GTA, which cannot serve as an initiation codon. Plasmids were constructed expressing either the wild-type repressor ORF or the ORFs containing the mutated GTA codons. When introduced into Pseudomonas aeruginosa, no immunity to superinfection by D3112 was observed when the second GTG had been mutated. Northern blotting analysis demonstrated that the D3112 c repressor is transcribed as a 900-nt mRNA. The promoter region was defined by transcriptional lacZ fusions and primer extension analyses to bp 972-940 from the left end of the phage genome. When the D3112 c repressor was overexpressed and purified as a fusion protein with a C-terminal six-histidine extension (cts15-His6), it showed high affinity for a 261-bp PvuII fragment localized directly upstream of the c repressor ORF. Our results indicate that although D3112 c shows higher amino acid similarity to the lambda family of repressors than it does to those of Mu and D108, it appears that its structure and function more accurately reflect an evolutionary ancestry with those from transposable coliphages Mu and D108., (Copyright 2000 Academic Press.)

The lytic-lysogenic switch in transposable, Mu-like bacteriophage D108 is governed by two divergent and slightly overlapping transcription units originating from the Pe and Pc promoters. DNase I footprinting and in vivo mutational analysis suggest that lysogeny is maintained by c-repressor occupancy of the O2 operator, which precludes RNA polymerase from binding to Pe. Lytic development is controlled by the Ner repressor, which binds to a site symmetrically situated between the converging promoters and, in the absence of other factors, prevents RNA polymerase from binding to either Pc or Pe. DNase I protection and potassium permanganate hypersensitivity in the presence of integration host factor (IHF), which binds and alters the DNA structure upstream of Pe, revealed that RNA polymerase was able to bind Pe irrespective of the Ner.DNA-bound complex, and partially unwind the Pe "-10 region." Ner repression of Pe transcription in vitro was significantly more effective in the absence of IHF. Using a cloned D108 early region-lacZ fusion in IHF-deficient and -proficient backgrounds, we also demonstrate this host factor's affect on ner-repressed Pe in vivo, and generate a system for isolating mutants in the regulatory genes and sites controlling this genetic switch. D108 lytic growth is proposed to occur through IHF-mediated activation of the phage Ner-repressed early operon.

From the transposable Mu-like bacteriophage D108 we have cloned the ner gene under the control of the lac UV5 promoter in the expression vector pOP95-15. The recombinant plasmid, pPT011, overproduced the 8-kd D108 ner protein (visualized by in vitro-coupled transcription-translation) and served as a substrate for DNA sequencing of the D108 ner gene. The ner protein of D108 was found to be 48% homologous to the Mu ner protein, though the DNA sequences that encode these proteins are quite divergent. We used the retardation of migration of 32P-labelled DNA restriction fragments by ner-containing crude protein extracts in polyacrylamide gels (band competition assay) to determine which DNA restriction fragment(s) contained the ner-binding sites. DNA footprinting using crude extracts physically identified the 47-bp DNA sequence that the ner protein was interacting with in the D108 early gene regulatory region. This sequence is located 10 bp downstream from the presumed D108 early gene transcription initiation site. Therefore, by binding strongly to this 47-bp DNA sequence, the D108 ner protein can regulate D108 early gene transcription.

The bacteriophage Mu ner gene has been cloned under the control of the lacUV5 promoter in the expression vector pOP95-15. The gene products of the recombinant plasmid, pUD88, visualized by in vitro coupled transcription-translation, are the bacteriophage Mu ner protein (8 kDa) and a 23 KDa protein consisting of the amino terminus of gpA (Mu transposase) fused to the carboxy terminus of beta-lactamase. DNA-binding activity was measured by the retardation of migration of a 32P-labeled DNA restriction fragment (containing the presumed ner-binding sites) in polyacrylamide gels. We have demonstrated specific association of ner to its binding sites to occur within 30 sec after the addition of impure extracts of ner overproducing cells. Much of this binding was dissociated within 30 sec by competition with a 20-fold molar excess of specific unlabeled DNA restriction fragment, but was resistant to dissociation when competed with unlabeled heterologous DNA for as long as 45 min at 37 degrees. By adapting a method for DNA-footprinting using impure extracts of ner overproducing cells, we were able to determine that the ner-binding sites are located between nucleotides 1026 and 1058 from the Mu left end. These results support the hypothesis that ner is similar to the cro regulatory protein from bacteriophage lambda and acts to regulate Mu early gene expression and the choice between lytic and lysogenic development.

We have cloned and characterized the right end terminal 796 bp of the transposable Mu-like bacteriophage D108. This region encompasses a 520 bp region of D108-specific sequences not present in phage Mu that contain an open reading frame encoding a 12 KDa protein. This protein can be visualized in vivo when the region is placed downstream from the strong lac UV5 promoter. The open reading frame can be expressed from the dam-regulated mod promoter (for modification of D108 DNA), yet also contains its own dam-independent promoter for expression that is detectable by northern blot analysis late in the D108 lytic cycle. Comparison of this region of D108 DNA with the corresponding region of Mu DNA suggests that a complex rearrangement has occurred at the phages' right ends during their evolution.

The 37 kb transposable bacteriophage Mu genome encodes a transposase protein which can recognize and bind to a consensus sequence repeated three times at each extremity of its genome. A subset of this consensus sequence (5'-PuCGAAA(A)-3') is found in the ends of many class II prokaryotic transposable elements. These elements, like phage Mu, cause 5 bp duplications at the site of element insertion, and transpose by a cointegrate mechanism. Using the band retardation assay, we have found that crude protein extracts containing overexpressed Mu transposase can form high-affinity protein-DNA complexes with Mu att R and the ends of the class II elements Tn 3 (right) and IS101. No significant protein-DNA complex formation was observed with DNA fragments containing the right end of the element IS102, or a non-specific pBR322 fragment of similar size. These results suggest that the Mu transposase protein can specifically recognize the ends of other class II transposable elements and that these elements may be evolutionarily related.

We have localized the D108 thermosensitive (cts) repressor gene to a region of DNA approx. 600 base pairs (bp) in length by sub-cloning an RsaI restriction endonuclease fragment (bp 200 to bp 802 from the left-end of the D108 genome). We determined that the gene product from this fragment appears to be the same size (19 kDa) as that expressed from clones containing larger fragments of D108 DNA. Results from in vitro gel electrophoresis band-retardation and in vivo immunity assays show that the sub-cloned repressor appears to be fully functional.

The temperate, transposable bacteriophages D108 and Mu are highly homologous, but differ in their lef-end regulatory regions. We have previously cloned the gene encoding the D108 thermo-sensitive (cts) repressor under the control of the lactUV5 promoter. In this work, we report that crude protein extracts containing highly-expressed D108 repressor protect a 77 bp region of DNA, located between 863 bp and 940 bp from the D108 lef--end, from both exonuclease III and DNase I hydrolysis. Nucleotide sequence analysis of this region reveals that is also contains DNA sequences homologous to the consensus DNA-binding site of the Escherichia coli protein, Integration Host Factor (IHF). Crude protein extracts containing highly-expressed IHF specifically bind to, and retard the migration of, DNA fragments containing the D108 regulatory region, and the DNA sequence which IHF protects from DNase I cleave lies directly within the D108 repressor binding region. There are two apparent repressor-specific S1 nuclease-resistant RNA suggests that transcription from the early region promoter, Pe may initiate at or about 1000 bp from the left-end of the D108 genome. Thus though, D108 and Mu utilize three analogous proteins (repressor, ner, and IHF) and the same apparent promoters for early gene regulation and the lytic/lysogenic decision, the organization of these regulatory components is apparently different, suggesting different mechanisms of control of gene expression.