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

Abstract

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°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 I-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.The outcome of an infection with bacteriophage Mu is either killing of the host cell or stable integration of phage DNA into the host chromosome and lysogenic growth of the bacterium. The decision that the virus must make to develop along one of these two pathways is complex (5, 28) and can be altered by conditions such as the multiplicity of infection and the genetic composition of the host cell (3,4,8,27). Two viral elements that are critically involved in the lysis-lysogeny decision are the Mu c repressor and its operator site located about 1 kb from the left end of viral DNA.The Mu operator is a large regulatory DNA sequence containing convergent promoters and sites for binding both phage-encoded and host-derived proteins. The two principal promoters that regulate phage development in the lytic and lysogenic pathways are PE and PCM. PE is an early promoter and the control point of a polycistronic operon encoding transposition and regulatory functions (20). The PcM promoter is the start point for a monocistronic transcript that proceeds toward the left end of the phage genome and encodes the repressor (11,18,19). The PE and PcM promoters are regulated by the Mu repressor, which binds in a cooperative manner to nearly 200 bp of operator DNA. The Mu regulatory region is organized into three blocks of DNA sequence designated operator sites 01, 02, and 03 (18, 19). Each operator site contains two or more 14-bp repressor consensus sites; there are 3, 4, and 2 consensus sequences localized in operator sites 01, 02, and 03, respectively (Fig. 1). An integration host factor binding site separates 01 and 02.The Mu repressor is composed of 22,000-Da protomers that oligomerize. At its N terminus, it contains significant structural homology with the N-terminal domain of Mu * Corresponding author. transposase (13) (Fig. 1). Interactions between repressor and supercoiled operator DNA have been modeled in vitro, and the following regulatory pattern was observed. As repressor concentrations were gradually increased, protection from DNase I cleavage was found in 01 and 02 and PE transcription gradu...

trans-Targeting of the Phage Mu Repressor Is Promoted by Conformational Changes That Expose Its ClpX Recognition Determinant

“…Construction of plasmids pGTN204 (RepC197) and pGTN211 (VirC192), derivatives of pREP-1 (31), and pGTN210 (Rep⌬18), a derivative of pHS502, were described previously (34). RepNK was overproduced from pREP-NK, which was constructed by PCR amplification of the c ϩ gene in pJV300 (38), using an N-terminal primer that contains the coding sequence for the cAMPdependent kinase motif (39). The primer adds the sequence GGACT-TCGAAGAGCTTCTGTTATC encoding the peptides GIRRASVI between the first two codons (Met 1 and Lys 2 ) of the repressor gene.…”

“…The leucine-rich domain located between the DBD and the CTD is hypothesized to play a role in repressor oligomerization (38) and is required for repressor activity (19,47,48). B, repressor proteins with a single cysteine at the C terminus for the attachment of MIANS.…”

The tRNA function of SsrA contributes to controlling repression of bacteriophage Mu prophage

“…Repcts62), and after 48 h of incubation at 30°C (S derepression) (11,13). The lysogens received the ssrA::Cm allele by bacteriophage P1 transduction and were transformed with plasmid pJW28, pJW30, pJW29, or pJW34, which express the WT, ssrA DD , ssrA 0 and ssrA UG , RNA, respectively.…”

“…We therefore analyzed Mu repressor by Western blotting in strains carrying the same ssrA alleles as above. As the protein could not be reliably detected in strains carrying a single copy of the Mu c gene, we used plasmid constructs of the pJV300 series (11). These pRS551 derivatives (based on the pBR322 replicon) carry the same Mu fragment as the JV phages described above, providing autoregulated expression of various forms of Mu repressor from the pCM promoter.…”

“…Four point mutations in the N-terminal part of Repc (cts45:S18L; cts71:M28I; cts25:D43G; and cts62:R47Q) (Fig. 1B) render the protein thermosensitive for operator binding, and Mucts lysogens thus become inducible at 42°C (10)(11)(12). The Mu prophage is also derepressed in stationary phase (called ''S'' derepression in this paper and refs.…”