Showing papers on "Lambda phage published in 2008"

Efficient point mutagenesis in mycobacteria using single-stranded DNA recombineering: Characterization of antimycobacterial drug targets

Abstract: Construction of genetically isogenic strains of mycobacteria is complicated by poor recombination rates and the lack of generalized transducing phages for Mycobacterium tuberculosis. We report here a powerful method for introducing single point mutations into mycobacterial genomes using oligonucleotide-derived single-stranded DNA recombineering and mycobacteriophage-encoded proteins. Phage Che9c gp61-mediated recombination is sufficiently efficient that single base changes can be introduced without requirement for direct selection, with isogenic mutant strains identified simply by PCR. Efficient recombination requires only short (50 nucleotide) oligonucleotides, but there is an unusually strong strand bias and an oligonucleotide targeting lagging strand DNA synthesis can recombine more than 10,000-fold efficiently than its complementary oligonucleotide. This ssDNA recombineering provides a simple assay for comparing the activities of related phage recombinases, and we find that both Escherichia coli RecET and phage lambda Red recombination proteins function inefficiently in mycobacteria, illustrating the utility of developing recombineering in new bacterial systems using host-specific bacteriophage recombinases. ssDNA mycobacterial recombineering provides a simple approach to characterizing antimycobacterial drug targets, and we have constructed and characterized single point mutations that confer resistance to isoniazid, rifampicin, ofloxacin and streptomycin.

Nongenetic Individuality in the Host–Phage Interaction

Abstract: Isogenic bacteria can exhibit a range of phenotypes, even in homogeneous environmental conditions. Such nongenetic individuality has been observed in a wide range of biological processes, including differentiation and stress response. A striking example is the heterogeneous response of bacteria to antibiotics, whereby a small fraction of drug-sensitive bacteria can persist under extensive antibiotic treatments. We have previously shown that persistent bacteria enter a phenotypic state, identified by slow growth or dormancy, which protects them from the lethal action of antibiotics. Here, we studied the effect of persistence on the interaction between Escherichia coli and phage lambda. We used long-term time-lapse microscopy to follow the expression of green fluorescent protein (GFP) under the phage lytic promoter, as well as cellular fate, in single infected bacteria. Intriguingly, we found that, whereas persistent bacteria are protected from prophage induction, they are not protected from lytic infection. Quantitative analysis of gene expression reveals that the expression of lytic genes is suppressed in persistent bacteria. However, when persistent bacteria switch to normal growth, the infecting phage resumes the process of gene expression, ultimately causing cell lysis. Using mathematical models for these two host–phage interactions, we found that the bacteria's nongenetic individuality can significantly affect the population dynamics, and might be relevant for understanding the coevolution of bacterial hosts and phages.

Bacteriophage infection is targeted to cellular poles.

Abstract: The poles of bacteria exhibit several specialized functions related to the mobilization of DNA and certain proteins. To monitor the infection of Escherichia coli cells by light microscopy, we developed procedures for the tagging of mature bacteriophages with quantum dots. Surprisingly, most of the infecting phages were found attached to the bacterial poles. This was true for a number of temperate and virulent phages of E. coli that use widely different receptors and for phages infecting Yersinia pseudotuberculosis and Vibrio cholerae. The infecting phages colocalized with the polar protein marker IcsA-GFP. ManY, an E. coli protein that is required for phage lambda DNA injection, was found to localize to the bacterial poles as well. Furthermore, labelling of lambda DNA during infection revealed that it is injected and replicated at the polar region of infection. The evolutionary benefits that lead to this remarkable preference for polar infections may be related to lambda's developmental decision as well as to the function of poles in the ability of bacterial cells to communicate with their environment and in gene regulation.

Crystal structure of the λ repressor and a model for pairwise cooperative operator binding

Abstract: A crystal structure of the intact, dimeric phage λ cI repressor bound to a DNA operator site reveals an unusual architecture allowing the repressor to adopt a conformation that facilitates cooperative binding to adjacent operator sites. Bacteriophage λ has for many years been a model system for understanding mechanisms of gene regulation1. A ‘genetic switch’ enables the phage to transition from lysogenic growth to lytic development when triggered by specific environmental conditions. The key component of the switch is the cI repressor, which binds to two sets of three operator sites on the λ chromosome that are separated by about 2,400 base pairs (bp)2,3. A hallmark of the λ system is the pairwise cooperativity of repressor binding4. In the absence of detailed structural information, it has been difficult to understand fully how repressor molecules establish the cooperativity complex. Here we present the X-ray crystal structure of the intact λ cI repressor dimer bound to a DNA operator site. The structure of the repressor, determined by multiple isomorphous replacement methods, reveals an unusual overall architecture that allows it to adopt a conformation that appears to facilitate pairwise cooperative binding to adjacent operator sites.

DNA looping can enhance lysogenic CI transcription in phage lambda

Abstract: The lysogenic state of bacteriophage lambda is maintained by CI repressor, which negatively regulates two promoters to block lytic gene expression. Expression of CI is itself controlled by positive and negative feedback as CI binds to O(R) to regulate the P(RM) promoter. In addition to direct interactions with operator DNA, CI tetramers bound at O(L) and O(R) can come together to form an octamer, looping the DNA that lies between them and allowing O(L) to assist with negative regulation of P(RM). We used a fluorescent reporter protein to measure the CI concentration for a set of constructs that differ in their ability to assume various forms of the looped structure. Based on the observed steady-state fluorescence for these constructs, the presence of O(L) increases P(RM) activation unless both operators can be fully occupied. By calculating the probabilities for the underlying operator configurations present in each construct, two different models for the mechanism of enhanced activation allow us to predict that when the DNA is looped, P(RM) activation can be 2- to 4-fold higher than is possible for unlooped DNA. Based on our results, transcriptional regulation for lambda's lysogenic/lytic switch includes both activation and repression due to DNA looping.

Host responses influence on the induction of lambda prophage.

Abstract: Inactivation of bacteriophage lambda CI repressor leads almost exclusively to lytic development. Prophage induction can be initiated either by DNA damage or by heat treatment of a temperature-sensitive repressor. These two treatments also cause a concurrent activation of either the host SOS or heat-shock stress responses respectively. We studied the effects of these two methods of induction on the lytic pathway by monitoring the activation of different lambda promoters, and found that the lambda genetic network co-ordinates information from the host stress response networks. Our results show that the function of the CII transcriptional activator, which facilitates the lysogenic developmental pathway, is not observed following either method of induction. Mutations in the cro gene restore the CII function irrespective of the induction method. Deletion of the heat-shock protease gene ftsH can also restore CII function following heat induction but not following SOS induction. Our findings highlight the importance of the elimination of CII function during induction as a way to ensure an efficient lytic outcome. We also show that, despite the common inhibitory effect on CII function, there are significant differences in the heat- and SOS-induced pathways leading to the lytic cascade.

Generation of safety enhanced Edwardsiella tarda ghost vaccine.

Abstract: A dual vector expressing the ghost-inducing PhiX174 lysis E gene and the bacterial DNA degrading staphylococcal nuclease A (SNA) gene was constructed to solve the problem of remnant antibiotic resistance genes and genomic DNA with intact pathogenic islands in the final product of Edwardsiella tarda ghosts (ETG). The SNA (devoid of secretion signal sequence and the nuclease B amino terminus sequence), fused with the 26 amino acid N-terminal sequence of the lambda phage Cro gene, showed successful degradation of bacterial nucleic acids. Furthermore, the nuclease activity of SNA in E. tarda was enhanced by codon optimization of the SNA gene using site-directed mutagenesis. ETG were generated via coexpression of the SNA gene and lysis gene E under the control of each lambdaP(R) promoter. The ghost bacteria generation system we describe is advantageous as it allows the use of a single plasmid, improves safety and vaccine purity by limiting residual genetic content from the ghost bacteria, and reduces production costs through cheap means of induction that use only temperature shifts.

Analysing Formal Models of Genetic Regulatory Networks with Delays: Applications to Lambda phage and T-cell Activation Systems

Abstract: In this paper, we propose a refinement of the modelling of biological regulatory networks based on the discrete approach of Rene Thomas. We refine and automatise the use of delays of activation/inhibition in order to specify which variable is more quickly affected by a change of its regulators. The formalism of linear hybrid automata is well suited to allow such refinement. We then use HyTech for two purposes: to find automatically all paths from a specified initial state to another one; to synthesise constraints on the delay parameters in order to follow any specific path.

A phage display system designed to detect and study protein-protein interactions.

Abstract: Analysing protein-protein interactions is critical in proteomics and drug discovery. The usage of 2-Hybrid (2lambda) systems is limited to an in vivo environment. We describe a bacteriophage 2-Hybrid system for studying protein interactions in vitro. Bait and prey are displayed as fusions to the surface of phage lambda that are marked with different selectable drug-resistant markers. An interaction of phages in vitro through displayed proteins allows bacterial infection by two phages resulting in double drug-resistant bacterial colonies at very low multiplicity of infections. We demonstrate interaction of the protein sorting signal Ubiquitin with the Vps9-CUE, a Ubiquitin binding domain, and by the interaction of (Gly-Glu)(4) and (Gly-Arg)(4) peptides. Interruptions of the phage interactions by non-fused (free) bait or prey molecules show how robust and unique our approach is. We also demonstrate the use of Ubiquitin and CUE display phages to find binding partners in a lambda-display library. The unique usefulness to 2lambda is also described.

Extended Function of Plasmid Partition Genes: the Sop System of Linear Phage-Plasmid N15 Facilitates Late Gene Expression

Abstract: The mitotic stability of the linear plasmid-prophage N15 of Escherichia coli depends on a partition system closely related to that of the F plasmid SopABC. The two Sop systems are distinguished mainly by the arrangement of their centromeric SopB-binding sites, clustered in F (sopC) and dispersed in N15 (IR1 to IR4). Because two of the N15 inverted repeat (IR) sites are located close to elements presumed (by analogy with phage lambda) to regulate late gene expression during the lytic growth of N15, we asked whether Sop partition functions play a role in this process. In N15, a putative Q antiterminator gene is located 6 kb upstream of the probable major late promoter and two intrinsic terminator-like sequences, in contrast to lambda, where the Q gene is adjacent to the late promoter. Northern hybridization and lacZ reporter activity confirmed the identity of the N15 late promoter (p52), demonstrated antiterminator activity of the Q analogue, and located terminator sequences between p52 and the first open reading frame. Following prophage induction, N15 mutated in IR2 (downstream from gene Q) or IR3 (upstream of p52) showed a pronounced delay in lysis relative to that for wild-type N15. Expression of ir3(-)-p52::lacZ during N15 wild-type lytic growth was strongly reduced relative to the equivalent ir3(+) fusion. The provision of Q protein and the IR2 and SopAB proteins in trans to ir3(+)-p52::lacZ increased expression beyond that seen in the absence of any one of these factors. These results indicate that the N15 Sop system has a dual role: partition and regulation of late gene transcription during lytic growth.