Chapter 27 Phage Strategies 27 1 Introduction bacteriophage

Chapter 27 Phage Strategies

27. 1 Introduction • bacteriophage (or phage) – A bacterial virus. • lytic infection – Infection of a bacterium by a phage that ends in the destruction of the bacterium with release of progeny phage. • lysis – The death of bacteria at the end of a phage infective cycle when they burst open to release the progeny of an infecting phage (because phage enzymes disrupt the bacterium’s cytoplasmic membrane or cell wall).

27. 1 Introduction FIGURE 01: A phage may follow the lytic or lysogenic pathway

27. 1 Introduction • virulent phage – A bacteriophage that can only follow the lytic cycle. • prophage – A phage genome covalently integrated as a linear part of the bacterial chromosome. • lysogeny – The ability of a phage to survive in a bacterium as a stable prophage component of the bacterial genome.

27. 1 Introduction • temperate phage – A bacteriophage that can follow the lytic or lysogenic pathway. • integration – Insertion of a viral or another DNA sequence into a host genome as a region covalently linked on either side to the host sequences. • excision – Release of phage from the host chromosome as an autonomous DNA molecule.

27. 1 Introduction • induction of phage – A phage’s entry into the lytic (infective) cycle as a result of destruction of the lysogenic repressor, which leads to excision of free phage DNA from the bacterial chromosome. • plasmid – Circular, extrachromosomal DNA. It is autonomous and can replicate itself. • episome – A plasmid able to integrate into bacterial DNA.

27. 2 Lytic Development Is Divided into Two Periods • A phage infective cycle is divided into the early period (before replication) and the late period (after the onset of replication). • A phage infection generates a pool of progeny phage genomes that replicate and recombine. FIGURE 02: Phages reproduce in lytic development

27. 3 Lytic Development Is Controlled by a Cascade • cascade – A sequence of events, each of which is stimulated by the previous one. – Transcriptional regulation is divided into stages, and at each stage one of the genes that is expressed encodes a regulator needed to express the genes of the next stage. FIGURE 03: Lytic development is a regulatory cascade

27. 3 Lytic Development Is Controlled by a Cascade • The early (or immediate early) genes transcribed by host RNA polymerase following infection include, or comprise, regulators required for expression of the middle (or delayed early) set of phage genes. • The middle group of genes includes regulators to transcribe the late genes. • This results in the ordered expression of groups of genes during phage infection.

27. 4 Two Types of Regulatory Events Control the Lytic Cascade • Regulator proteins used in phage cascades may sponsor initiation at new (phage) promoters or cause the host polymerase to read through transcription terminators. FIGURE 06: RNA polymerase controls promoter recognition.

27. 5 The Phage T 7 and T 4 Genomes Show Functional Clustering • Genes concerned with related functions are often clustered. FIGURE 08: T 4 genes show functional clustering

27. 5 The Phage T 7 and T 4 Genomes Show Functional Clustering • Phages T 7 and T 4 are examples of regulatory cascades in which phage infection is divided into three periods. FIGURE 09: T 4 genes fall into two general groups

27. 6 Lambda Immediate Early and Delayed Early Genes Are Needed for Both Lysogeny and the Lytic Cycle • Lambda has two immediate early genes, N and cro, which are transcribed by host RNA polymerase. • The N gene is required to express the delayed early genes. • Three of the delayed early genes are regulators.

27. 6 Lambda Immediate Early and Delayed Early Genes Are Needed for Both Lysogeny and the Lytic Cycle • Lysogeny requires the delayed early genes c. II-c. III. • The lytic cycle requires the immediate early gene cro and the delayed early gene Q. FIGURE 10: Lambda has two lifestyles

27. 7 The Lytic Cycle Depends on Antitermination by p. N • p. N is an antitermination factor that allows RNA polymerase to continue transcription past the ends of the two immediate early genes. • p. Q is the product of a delayed early gene and is an antiterminator that allows RNA polymerase to transcribe the late genes. FIGURE 12: Similar controls apply to left and right transcription

27. 7 The Lytic Cycle Depends on Antitermination by p. N • Lambda DNA circularizes after infection; as a result, the late genes form a single transcription unit. FIGURE 13: Lambda has three stages of development

27. 8 Lysogeny Is Maintained by the Lambda Repressor Protein • The lambda repressor, encoded by the c. I gene, is required to maintain lysogeny. • The lambda repressor acts at the OL and OR operators to block transcription of the immediate early genes. • The immediate early genes trigger a regulatory cascade; as a result, their repression prevents the lytic cycle from proceeding. FIGURE 15: Repressor maintains lysogeny

27. 9 The Lambda Repressor and Its Operators Define the Immunity Region • immunity – In phages, the ability of a prophage to prevent another phage of the same type from infecting a cell. • virulent mutations – Phage mutants that are unable to establish lysogeny.

27. 9 The Lambda Repressor and Its Operators Define the Immunity Region • Several lambdoid phages have different immunity regions. • A lysogenic phage confers immunity to further infection by any other phage with the same immunity region. FIGURE 16: RNA polymerase initiates at Pl and Pr but not at Prm during the lytic cycle.

27. 10 The DNA-Binding Form of the Lambda Repressor Is a Dimer • • A repressor monomer has two distinct domains. The N-terminal domain contains the DNA-binding site. The C-terminal domain dimerizes. Binding to the operator requires the dimeric form so that two DNA-binding domains can contact the operator simultaneously.

27. 10 The DNA-Binding Form of the Lambda Repressor Is a Dimer • Cleavage of the repressor between the two domains reduces the affinity for the operator and induces a lytic cycle. FIGURE 18: Repressor cleavage induces lytic cycle

27. 11 Lambda Repressor Uses a Helix-Turn -Helix Motif to Bind DNA • Each DNA-binding region in the repressor contacts a half -site in the DNA. • The DNA-binding site of the repressor includes two short α-helical regions that fit into the successive turns of the major groove of DNA (helix-turn-helix). • A DNA-binding site is a (partially) palindromic sequence of 17 bp. FIGURE 19: The operator is a palindrome

27. 11 Lambda Repressor Uses a Helix-Turn -Helix Motif to Bind DNA • The amino acid sequence of the recognition helix makes contacts with particular bases in the operator sequence that it recognizes. FIGURE 22: Helix-3 determines DNA-binding specificity

27. 12 Lambda Repressor Dimers Bind Cooperatively to the Operator • Repressor binding to one operator increases the affinity for binding a second repressor dimer to the adjacent operator. • The affinity is 10× greater for OL 1 and OR 1 than other operators, so they are bound first. • Cooperativity allows repressor to bind the OL 2/OR 2 sites at lower concentrations. FIGURE 25: Lambda repressors bind DNA cooperatively

27. 13 Lambda Repressor Maintains an Autoregulatory Circuit • The DNA-binding region of repressor at OR 2 contacts RNA polymerase and stabilizes its binding to PRM. • This is the basis for the autoregulatory control of repressor maintenance. • Repressor binding at OL blocks transcription of gene N from PL. FIGURE 26: Repressor maintains lysogeny but is absent during the lytic cycle

27. 13 Lambda Repressor Maintains an Autoregulatory Circuit • Repressor binding at OR blocks transcription of cro, but also is required for transcription of c. I. • Repressor binding to the operators therefore simultaneously blocks entry to the lytic cycle and promotes its own synthesis. FIGURE 27: Helix-2 interacts with DNA polymerase

27. 14 Cooperative Interactions Increase the Sensitivity of Regulation • Repressor dimers bound at OL 1 and OL 2 interact with dimers bound at OR 1 and OR 2 to form octamers. • These cooperative interactions increase the sensitivity of regulation. FIGURE 29: Repressors to bind to OL 3 and OR 3 at higher concentrations

27. 15 The c. II and c. III Genes Are Needed to Establish Lysogeny • The delayed early gene products c. II and c. III are necessary for RNA polymerase to initiate transcription at the promoter PRE. • c. II acts directly at the promoter and c. III protects c. II from degradation. • Transcription from PRE leads to synthesis of repressor and also blocks the transcription of cro. FIGURE 30: Repressor establishment uses a special promoter

27. 16 A Poor Promoter Requires c. II Protein • PRE has atypical sequences at – 10 and – 35. • RNA polymerase binds the promoter only in the presence of c. II. • c. II binds to sequences close to the – 35 region. FIGURE 31: c. II enables RNA polymerase to bind to PRE

27. 17 Lysogeny Requires Several Events • c. II and c. III cause repressor synthesis to be established and also trigger inhibition of late gene transcription. • Establishment of repressor turns off immediate and delayed early gene expression. • Repressor turns on the maintenance circuit for its own synthesis. • Lambda DNA is integrated into the bacterial genome at the final stage in establishing lysogeny.

27. 17 Lysogeny Requires Several Events FIGURE 33: The lysogenic pathway leads to repressor synthesis

27. 18 The Cro Repressor Is Needed for Lytic Infection • Cro binds to the same operators as the lambda repressor, but with different affinities. • When Cro binds to OR 3, it prevents RNA polymerase from binding to PRM and blocks the maintenance of repressor promoter.

27. 18 The Cro Repressor Is Needed for Lytic Infection • When Cro binds to other operators at OR or OL, it prevents RNA polymerase from expressing immediate early genes, which (indirectly) blocks repressor establishment. FIGURE 34: The lytic pathway leads to expression of cro and late genes

27. 19 What Determines the Balance between Lysogeny and the Lytic Cycle? • The delayed early stage when both Cro and repressor are being expressed is common to lysogeny and the lytic cycle. • The critical event is whether c. II causes sufficient synthesis of repressor to overcome the action of Cro.

27. 19 What Determines the Balance between Lysogeny and the Lytic Cycle? FIGURE 35: Repressor determines lysogeny, and Cro determines the lytic cycle