The bacteriophage D108 Ner repressor binds a conformationally distinct operator

The Ner protein encoded by the transposable coliphage D108, an 8.6 kDa λ Cro-like repressor, binds to an operator spanning 50 bp of DNA. The distinguishing features of this operator are two perfect 11-bp inverted repeats (5′-CCGTGAGCTAC-3′) that are separated by an 8-bp AT-rich spacer. Hyperreactivity of the ner operator to potassium permanganate and the hydroxyl radical indicate that the AT-rich spacer assumes a variant conformation consistent with a bend. Using an electrophoretic mobility shift assay, we demonstrated that Ner does not display significant affinity for a single 11-bp site. Furthermore, DNase I protection analysis and circular-permutation binding assays reveal that alterations in the length and sequence of the AT-rich spacer that separates the 11-bp inverted repeats significantly alter Ner-operator interactions, and demonstrate that the intrinsically bent ner operator is conformationally altered upon protein binding. Received: 29 September 1999 / Accepted: 21 December 1999     

Genetic characterization of Mu-like bacteriophage D108.

Infection of Escherichia coli by bacteriophage D108 was shown to result in the generation of apparently random chromosomal mutations. Approximately 1% of the cells lysogenized by D108, as with Mu, acquired new auxotrophic mutations. D108-induced mutations were nonreverting and were most probably the result of insertion of the D108 genome into regions of genetic function. D108 and Mu shared many similar properties but were heteroimmune and had different host ranges. Lytic infections of Mu lysogens with D108 and D108 lysogens with Mu resulted in 100-fold increases in release of phage with prophage markers over those due to spontaneous induction. Phenotypic mixing was common, with most phage carrying the prophage immunity being packaged in particles with the host range of the superinfecting phage. A fraction of the superinfecting phage genomes were, however, packaged in particles with the prophage-specified host range. Although 10% of the prophage progeny were D108-Mu genetic hybrids, superinfecting phage-induced release of the prophage with reciprocal phenotypic mixing occurred in recA hosts, in which the frequency of D108-Mu genetic hybrids was reduced 100-fold.     

Purification and characterization of the Ner repressor of bacteriophage Mu

The Ner protein of bacteriophage Mu acts as a lambda cro-like negative regulator of the phage's early (transposase) operon. Using the band retardation assay to monitor ner-operator-specific DNA-binding activity, the 8 kDa Ner protein was purified to homogeneity. DNase I footprinting revealed that the purified protein bound and protected a specific DNA operator that contains two 12 bp sites with the consensus sequence 5'-ANPyTAPuCTAAGT-3', separated by a 6 bp spacer region. Moreover, regions corresponding to a turn of the DNA helix flanking these 12 bp repeats are also protected by Ner. Unlike the functionally similar lambda cro protein, gel filtration experiments show that the native molecular mass of Mu Ner to be approx. 8 kDa. These results, plus the pattern of DNase I protection, suggest that the protein may bind as a monomer to each of its specific DNA substrates.     

Cloning and localization of the repressor gene (c) of the Mu-like transposable phage D108

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.     

Chromosomal rearrangements induced by temperate bacteriophage D108.

As previously shown for mutator phage Mu-1, to which it is closely related, temperate bacteriophage D108 induces chromosomal rearrangements (replicon fusion and transposition of chromosomal segments) in its host genome.     

Purification and characterization of the DNA-binding protein Ner of bacteriophage Mu

The construction is described of a plasmid (pL-ner) which directs the high-level production of the bacteriophage Mu Ner protein in Escherichia coli. The protein, recovered in the soluble cellular fraction, was susceptible to in vivo proteolytic processing, in many host strains, but not in E. coli B, a natural lon- prototroph. A simple purification method is described which takes advantage of the basic nature of the protein. The purified protein was shown to be physically and chemically homogeneous and to have an amino acid sequence identical to that predicted for the authentic protein. The protein was also shown to have in vitro biological activity, as measured by specific binding to a DNA fragment containing the consensus Ner-binding sequence, and in vivo biological activity as the protein produced by the pL-ner plasmid allowed lysogenic-like maintenance of a Mu prophage c mutant unable to synthesise a functional Mu repressor.     

Identification of the bacteriophage D108 kil gene and of the second region of sequence nonhomology with bacteriophage Mu

To identify the second region of sequence nonhomology between the genomes of the transposable bacteriophages Mu and D108 originally observed by electron-microscopic analysis of DNA heteroduplexes and to localize functions ascribed to the 'accessory' or 'semi-essential' early regions of the phages between genes B and C, a 0.9-kb fragment of each genome located immediately beyond the B gene was cloned and sequenced. Three open reading frames (ORFs) were identified in each. The region of nonhomology is located within the 3' portion of the third ORF. D108 is shown to possess a Kil function similar to that previously shown for Mu, and that function is encoded by the first ORF.     

The right end of transposable bacteriophage D108 contains a 520 base pair protein-encoding sequence not present in bacteriophage Mu.

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.     

Mutator bacteriophage D108 and its DNA: an electron microscopic characterization

Three types of phage particles were observed on CsCl step gradients when D108 was purified from lysates prepared by induction of a prophage. These particle types were identified to be the mature phage, tailless DNA-filled heads, and a form of nucleoprotein aggregates. The nucleoprotein aggregates banded at a density (rho) of greater than 1.6. DNA molecules isolated from mature phage particles were (38.305 +/- 1.226) kilobases (kb) in length. Denaturation and renaturation of D108 DNA resulted in the formation of linear double-stranded molecules with variable-length single-stranded tails at one end. About 30% of the annealed molecules also carried an internal nonhomology, which was shown to be the region called the G-loop in Mu and P1 DNAs. Following the notation used for different regions of denatured, annealed Mu DNA, we measured the lengths of the equivalent D108 DNA regions to be as alpha-D108 = (32.178 +/- 1.370) kb; G-D108 = (3.07 +/- 0.382) kb; beta-D108 = (2.291 +/- 0.306) kb; SE-D108 = (0.966 +/- 0.433) kb. Formation of D108; Mu heteroduplexes disclosed the presence of five nonhomologies, two of which were partial. One of the partial heterologies was in the G-loop region. The largest nonhomology, (1.393 +/- 0.185) kb in size, was near the c end (immunity region) and probably spans the c and the ner genes of Mu. beta-D108 was shown to carry a (0.556 +/- 0.097)-kb insertion close to its right end. A short 100-base-pair region appeared to have been conserved at the ends of D108 and Mu. Occasionally, a 50-to 100-base-pair-long unpaired region was also observed at the left end of D108: Mu heteroduplexes. These sequences were presumably of bacterial DNA. Taken together, our results complement and extend our earlier genetic studies which established that D108 was a mutator phage heteroimmune to Mu with a host range different from Mu's.     

Comparison of the structures of cro and lambda repressor proteins from bacteriophage lambda

The three-dimensional structures of cro repressor protein and of the amino-terminal domain of lambda repressor protein, both from bacteriophage lambda, are compared. The second and third alpha-helices, alpha 2 and alpha 3, are shown to have essentially identical conformations in the two proteins, confirming the significance of the amino acid sequence homology previously noted between these and other DNA binding proteins in the region corresponding to these helices. The correspondence between the two-helical units in cro and lambda repressor protein is better than the striking agreement noted previously between two-helical units in cro and catabolite gene-activator protein. Parts of the first alpha-helices of repressor and cro show a structural correspondence that suggests a revised sequence homology between the two proteins in their extreme amino-terminal regions. In particular, there is a short loop between the alpha 1 and alpha 2 helices of lambda repressor that is missing from cro. This structural difference may account for the observed differences found with different cros and repressors in the pattern of phosphates whose ethylation prevents the binding of these proteins to their specific recognition sites. Although the two proteins have strikingly similar alpha 2-alpha 3 helical units that are presumed to bind to DNA in an essentially similar manner, stereochemical restrictions prevent the alpha 2-alpha 3 units of the respective proteins aligning on the DNA in exactly the same way.