Protein degradation in E. coli: The ion mutation and bacteriophage lambda N and cll protein stability

doi:10.1016/0092-8674(81)90518-3

Cell

Volume 24, Issue 1, April 1981, Pages 225-233

https://doi.org/10.1016/0092-8674(81)90518-3 Get rights and content

Abstract

The Ion gene of E. coli controls the stability of two bacteriophage lambda proteins. The functional half-life of the phage N gene product, measured by complementation, is increased about 5-fold in Ion mutant strains, from 2 min to 10 min. The chemical half-life of N protein, determined by its disappearance on polyacrylamide gels following pulse-chase labeling, increases about three-fold in Ion cells. In contrast to its effect on the N protein, the Ion mutation produces a 50% decrease in the chemical half-life of cll protein. The decay rate of many other phage proteins, including the unstable gene O product, remains unaffected by a host Ion defect. A Ion mutation alters lambda physiology in two ways. First, upon infection, the phage enters the lytic pathway predominantly. This may result from the deficiency of cll protein caused by its decreased stability, since cll product is required for establishment of lysogeny. Second, brief thermal induction of a Ion (λc1857) lysogen leads irreversibly to lysis; repression cannot be restablished and the treated cells are committed to forming infective centers. Although N product is normally required for rapid commitment, Ion lysogens become committed more rapidly than Ion⁺ lysogens, even in the absence of N function. These results identify for the first time native proteins whose stability is affected by the Lon proteolytic pathway. They also indicate that the Lon system may be important in regulating gene expression in E. coli.

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We speculate that Lon may play an essential role in the regulation of DNA replication depending on growth conditions. E. coli strains used in this study were BL21(DE3), C600, and its derivative ATC12017 (C600 lon510) (59, 60). Sequences of oligonucleotides used in this work are listed in supplemental Table S1: 1–6, primers for obtaining truncated mutants of Lon protein; 7–12, primers for site-directed mutagenesis to introduce substitutions in the ATPase domain of Lon protease; 13 and 14, primers for preparation of the EMSA probe; 15 and 16, complementary single-stranded oligonucleotides (Thermo Scientific) for obtaining the 5′-terminally biotinylated dsDNA fragment used for the SPR analysis.
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The Lon protease was the first ATP-dependent protease to be identified. It was evidenced as the product of the Lon gene in Escherichia coli and its implication in protein stability and abnormal protein degradation was first demonstrated in this bacteria (Gottesman et al., 1981; Chung and Goldberg, 1981). The human Lon was then identified as a mitochondrial protease by Wang et al. (1993).
Mitochondria have been implicated in the ageing process and the lifespan modulation of model organisms. Mitochondria are the main providers of energy in eukaryotic cells but also represent both a major source of reactive oxygen species and targets for protein oxidative damage. Since protein damage can impair mitochondrial function, mitochondrial proteases are critically important for protein maintenance and elimination of oxidized protein. In the mitochondrial matrix, protein quality control is mainly achieved by the Lon and Clp proteases which are also key players in damaged mitochondrial proteins degradation. Accumulation of damaged macromolecules resulting from oxidative stress and failure of protein maintenance constitutes a hallmark of cellular and organismal ageing and is believed to participate to the age-related decline of cellular function. Hence, age-related impairment of mitochondrial protein quality control may therefore contribute to the age-associated build-up of oxidized protein and alterations of mitochondrial redox and protein homeostasis.
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Thus, when their synthesis stops, their DNA replication initiation, delayed early gene expression and lysogeny promoting activities are quickly lost (Kobiler et al., 2004). The host proteases utilized for this purpose are ClpX/ClpP for O (Gonciarz-Swiatek et al., 1999; Thibault and Houry, 2012), Lon for N (Gottesman et al., 1981; Maurizi, 1987; Mikita et al., 2013) and FtsH for CII and CIII (Shotland et al., 2000; Kobiler et al., 2002, 2007), and Lon for Xis (Leffers and Gottesman, 1998). In addition to preventing the lytic gene transcription cascade, the CI protein has two other known regulatory functions, turning down expression of the host pckA gene and autoregulating its own synthesis.
Molecular genetic research on bacteriophage lambda carried out during its golden age from the mid-1950s to mid-1980s was critically important in the attainment of our current understanding of the sophisticated and complex mechanisms by which the expression of genes is controlled, of DNA virus assembly and of the molecular nature of lysogeny. The development of molecular cloning techniques, ironically instigated largely by phage lambda researchers, allowed many phage workers to switch their efforts to other biological systems. Nonetheless, since that time the ongoing study of lambda and its relatives has continued to give important new insights. In this review we give some relevant early history and describe recent developments in understanding the molecular biology of lambda׳s life cycle.
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Differential stability of toxins and antitoxins is the key for the conditional activation and function of Toxin–Antitoxin systems. Here we report the evaluation of the action of cell proteases Lon, ClpAP, ClpXP and ClpYQ on the Kis antitoxin and the Kid toxin of the parD TA system of plasmid R1. In vitro analysis shows that Kis antitoxin, but not the Kid toxin, is cleaved specifically by the ClpAP protease. The Kid toxin is not cleaved either by this protease or by any of the others cell proteases tested but in complex with the Kis antitoxin protects the cleavage of this protein in a way that is dependent on the toxin–antitoxin ratio. We further show that this protection is correlated with the inability of the ClpA chaperone to access the Kis antitoxin when in complex with Kid toxin. The stability of the antitoxin greatly increases in vivo in a clpP- background and plasmid maintenance mediated by the parD system, which is dependent on the differential decay of the antitoxin, is reduced to the levels observed in the absence of a functional toxin. The functional implications of these data are further discussed within the frame of the regulation of the parD system and of the available information on the nature of the toxin–antitoxin complexes formed at different toxin–antitoxin ratios.
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Bacteriophages T7, λ, P22, and P2/P4 (from Escherichia coli), as well as ϕ29 (from Bacillus subtilis), are among the best-studied bacterial viruses. This chapter summarizes published protein interaction data of intraviral protein interactions, as well as known phage–host protein interactions of these phages retrieved from the literature. We also review the published results of comprehensive protein interaction analyses of Pneumococcus phages Dp-1 and Cp-1, as well as coliphages λ and T7. For example, the ≈ 55 proteins encoded by the T7 genome are connected by ≈ 43 interactions with another ≈ 15 between the phage and its host. The chapter compiles published interactions for the well-studied phages λ (33 intra-phage/22 phage-host), P22 (38/9), P2/P4 (14/3), and ϕ29 (20/2). We discuss whether different interaction patterns reflect different phage lifestyles or whether they may be artifacts of sampling. Phages that infect the same host can interact with different host target proteins, as exemplified by E. coli phage λ and T7. Despite decades of intensive investigation, only a fraction of these phage interactomes are known. Technical limitations and a lack of depth in many studies explain the gaps in our knowledge. Strategies to complete current interactome maps are described. Although limited space precludes detailed overviews of phage molecular biology, this compilation will allow future studies to put interaction data into the context of phage biology.
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^∗: Present address: Department of Microbiology, Ohio State University, Columbus, Ohio 43210.

^†: Present address: Cancer Biology Program, Frederick Cancer Research Center, P.O. Box B, Frederick, Maryland 21701.

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Cell

ArticleProtein degradation in E. coli: The ion mutation and bacteriophage lambda N and cll protein stability

Abstract

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J. Mol. Biol.

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A mutant of Escherichia coli that is lysogenized with high frequency

The roles of the lambda clll gene and the Escherichia coli catabolite gene activation system in the establishment of lysogeny by bacteriophage lambda

Synthesis and segretion of secretory proteins: the signal hypothesis

Transfer of proteins across membranes. I. Presence of proteolytically processed and nonprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma

J. Cell. Biol.

Mutants on Escherichia coli with a defect in the degradation of nonsense fragments

Nature New Biol.

Isolation and characterization of deletions in bacteriophage λ residing as prophage in E. coli K12

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E. coli recA protein-directed cleavage of phage λ repressor requires polynucleotide

Nature

Mutations altering the cellular localization of the phage a receptor, an Escherichia coli outer membrane protein

Early protein synthesis and its control in bacteriophage lambda

Identification and mapping of the proteins encoded by the att-Q region of bacteriophage lambda

Gene

Gene Ion and plasmid inheritance in E. coli K12

J. Bacteriol.

Isolation of plaque-forming galactose-transducing strains of phage lambda

Genetics

Article
Protein degradation in E. coli: The ion mutation and bacteriophage lambda N and cll protein stability