No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Over-expression of rexA nullifies T4rII exclusion in Escherichia coli K(λ) lysogens
Abstract
Dosage and relative cellular levels of RexA and RexB proteins encoded by the rexA-rexB genes of a lambda prophage are important for the Rex+ phenotype, which was nullified when greater RexA or RexB was provided than was necessary for the complementation of a rexA- or a rexB- prophage.
... Rex exclusion of infecting T4rII is sensitive to the stoichiometric balance between rexA and rexB proteins. The overexpression of rexA relative to rexB nullifies Rex exclusion of infecting T4rII phage by E. coli K (λ) lysogens (Slavcev and Hayes 2004), and in the absence of a signal from an infecting phage the overexpression of rexA to rexB results in cellular growth arrest (Snyder and McWilliams 1989). Conversely, if rexB is overexpressed relative to rexA, then Rex exclusion is suppressed (Parma et al. 1992; Slavcev and Hayes 2004 ). ...
... The overexpression of rexA relative to rexB nullifies Rex exclusion of infecting T4rII phage by E. coli K (λ) lysogens (Slavcev and Hayes 2004), and in the absence of a signal from an infecting phage the overexpression of rexA to rexB results in cellular growth arrest (Snyder and McWilliams 1989). Conversely, if rexB is overexpressed relative to rexA, then Rex exclusion is suppressed (Parma et al. 1992; Slavcev and Hayes 2004 ). Rex exclusion was suggested to be " triggered " by some aspect of the replication of an infecting phage (Toothman and Herskowitz 1980b ), but Rex + activation and mechanism remain undetermined. ...
The cI-rexA-rexB operon of bacteriophage lambda confers 2 phenotypes, Imm and Rex, to lysogenic cells. Immunity to homoimmune infecting lambda phage depends upon the CI repressor. Rex exclusion of T4rII mutants requires RexA and RexB proteins. Both Imm and Rex share temperature-sensitive conditional phenotypes when expressed from cI[Ts]857 but not from cI+ lambda prophage. Plasmids were made in which cI-rexA-rexB was transcribed from a non-lambda promoter, pTet. The cI857-rexA-rexB plasmid exhibited Ts conditional Rex and CI phenotypes; the cI+-rexA-rexB plasmid did not. Polarity was observed within cI-rexA-rexB transcription at sites in cI and rexA when CI was nonfunctional. Renaturation of the Ts CI857 repressor, allowing it to regain functionality, suppressed the polar effect on downstream transcription from the site in cI. The second strong polar effect near the distal end of rexA was observed for transcription initiated from pE. The introduction of a rho Ts mutation into the host genome suppressed both polar effects, as measured by its suppression of the conditional Rex phenotype. Strong suppression of the conditional Rex[Ts] phenotype was imparted by ssrA and clpP (polar for clpX) null mutations, suggesting that RexA or RexB proteins made under conditions of polarity are subject to 10Sa RNA tagging and ClpXP degradation.
... Phages display a number of antiabortive infection mechanisms. In phage T4, for example, the rII genes are protective against the abortive infection mechanisms associated with the phage rex gene product of phage l lysogens (Slavcev and Hayes, 2004). Another well-studied abortive infection mechanism is that of phage T7 infecting F-plasmid-containing E. coli (Maekelae et al., 1964). ...
Host range describes the breadth of organisms a parasite is capable of infecting, with limits on host range stemming from parasite, host, or environmental characteristics. Parasites can adapt to overcome host or environmental limitations, while hosts can adapt to control the negative impact of parasites. We consider these adaptations as they occur among bacteriophages (phages) and their bacterial hosts, since they are significant to phage use as antibacterials (phage therapy) or to protection of industrial ferments from phage attack. Initially, we address how phage host range can (and should) be defined plus summarize claims of host ranges spanning multiple bacterial genera. Subsequently, we review bacterial mechanisms of phage resistance. These include adsorption resistance, which results in reduced interaction between phage and bacterium; what we describe as "restriction," where bacteria live but phages die; and abortive infections, where both phage and bacterium die. Adsorption resistance includes loss of phage receptor molecules on hosts as well as physical barriers hiding receptor molecules (e.g., capsules). Restriction mechanisms include phage-genome uptake blocks, superinfection immunity, restriction modification, and CRISPR, all of which function postphage adsorption but prior to terminal phage takeover of host metabolism. Standard laboratory selection methods, involving exposure of planktonic bacteria to high phage densities, tend to directly select for these prehost-takeover resistance mechanisms. Alternatively, resistance mechanisms that do not prevent bacterium death are less readily artificially selected. Contrasting especially bacteria mutation to adsorption resistance, these latter mechanisms likely are an underappreciated avenue of bacterial resistance to phage attack.
Bacteriophages are probably the oldest viruses, having appeared early during bacterial evolution. Therefore, bacteria and bacteriophages have a long history of co-evolution in which bacteria have developed multiple resistance mechanisms against bacteriophages. These mechanisms, that are very diverse and are in constant evolution, allow the survival of the bacteria. Bacteriophages have adapted to bacterial defense systems, devised strategies to evade these anti-phage mechanisms and restored their infective capacity. In this chapter, we review the bacterial strategies that hinder the phage infection as well as the counter-defense mechanisms developed.
T4 requires two proteins: holin, T (lesion formation and lysis timing) and endolysin, E (cell wall degradation) to lyse the host at the end of its life cycle. E is a cytoplasmic protein that sequestered away from its substrate, but the inner membrane lesion formed by T allows E to gain access to the cell wall. T4 exhibits lysis inhibition (LIN), a phenomenon in which a second T4 infection occurs ≤ 3 min after primary infection results a delay in lysis. Mutations that abolish LIN mapped to several genes but only rV encoding the holin, T, and rI, encoding the antiholin, RI, are required for LIN in all hosts which support T4 replication. Antiholin RI inhibits T-mediated lysis by direct interaction with the holin. T has at least one transmembrane domain with its Nterminus (TNTD) in the cytoplasm and C-terminus in the periplasm (TCTD). In contrast, the N-terminus of RI (RINTD) is predicted to function as a cleavable signal sequence allowing the secretion of the RI C-terminal domain (RICTD) into the periplasm. Most of RI mutations which abolish LIN occur in the RICTD, suggesting RI inhibits T-mediated lysis by interacting with T via RICTD. Topological analysis of RI and T showed that fusion of PhoA signal sequence (ssPhoA) to RICTD is necessary and sufficient for LIN and ssPhoAΦTCTD interferes with RI-mediated LIN, indicating T and RI interact via periplasmic C-terminal domains. In T4 infection, LIN is observed only when superinfection takes place, indicating either the antiholin or the LIN signal must be unstable. Both RI and RINTDΦPhoA are localized to both the inner membrane and the periplasm suggesting that the RINTD is a Signal-Anchor-Release (SAR) domain. Protein stability studies indicated that the SAR domain is the proteolytic determinant of RI, and DegP is the protease that is responsible for RI degradation. To date, how TNTD participates in lysis and LIN is not known. Modifications and deletion of the N-terminus of T change the lysis time, indicating this domain is involved the in timing of lysis. GFP fusion to holin T allowed microscopic visualization of fluorescent patches on the membrane at the time of lysis.
The rex operon of bacteriophage lambda excludes the development of several unrelated bacteriophages. Here we present an additional lambda rexB function: it prevents degradation of the short-lived protein lambda O known to be involved in lambda DNA replication. We have shown that it is the product of rexB that is responsible for the stabilization of lambda O: when a nonsense mutation is present in rexB, lambda O protein is labile; suppression of the mutation by the corresponding nonsense suppressor causes partial restabilization of lambda O. lambda rexB also stabilizes lambda O in trans. We discuss our results in relation to the function of rexB in lambda DNA replication and its role in the protein degradation pathways of bacteriophage lambda.
In Escherichia coli, programmed cell death is mediated through "addiction modules" consisting of two genes; the product of one gene is long-lived and toxic, whereas the product of the other is short-lived and antagonizes the toxic effect. Here we show that the product of lambdarexB, one of the few genes expressed in the lysogenic state of bacteriophage lambda, prevents cell death directed by each of two addiction modules, phd-doc of plasmid prophage P1 and the rel mazEF of E. coli, which is induced by the signal molecule guanosine 3',5'-bispyrophosphate (ppGpp) and thus by amino acid starvation. lambdaRexB inhibits the degradation of the antitoxic labile components Phd and MazE of these systems, which are substrates of ClpP proteases. We present a model for this anti-cell death effect of lambdaRexB through its action on the ClpP proteolytic subunit. We also propose that the lambdarex operon has an additional function to the well known phenomenon of exclusion of other phages; it can prevent the death of lysogenized cells under conditions of nutrient starvation. Thus, the rex operon may be considered as the "survival operon" of phage lambda.
We asked whether Rex exclusion encoded by a lambda prophage confers a protective or a cell-killing phenotype. We found that
the Rex system can channel lysogenic cells into an arrested growth phase that gives an overall protective ability to the host
despite some associated killing.
The rexA and rexB genes of bacteriophage lambda encode a two-component system that aborts lytic growth of bacterial viruses. Rex exclusion is characterized by termination of macromolecular synthesis, loss of active transport, the hydrolysis of ATP, and cell death. By analogy to colicins E1 and K, these results can be explained by depolarization of the cytoplasmic membrane. We have fractionated cells to determine the intracellular location of the RexB protein and made RexB-alkaline phosphatase fusions to analyze its membrane topology. The RexB protein appears to be a polytopic transmembrane protein. We suggest that RexB proteins form ion channels that, in response to lytic growth of bacteriophages, depolarize the cytoplasmic membrane. The Rex system requires a mechanism to prevent lambda itself from being excluded during lytic growth. We have determined that overexpression of RexB in lambda lysogens prevents the exclusion of both T4 rII mutants and lambda ren mutants. We suspect that overexpression of RexB is the basis for preventing self-exclusion following the induction of a lambda lysogen and that RexB overexpression is accomplished through transcriptional regulation.
The rexA and rexB genes of bacteriophage lambda are expressed from the prophage and cause the exclusion of many superinfecting mutant phages. We cloned the rexA and rexB genes into a multicopy plasmid so that they were overexpressed from the inducible tac promoter. No obvious phenotypes were associated with overexpressing both rexA and rexB or overexpressing rexA in the absence of rexB expression. However, induction of rexA in the presence of limiting rexB activity caused an immediate cessation of cell growth. All macromolecular synthesis abruptly ceased and amino acid transport was severely inhibited. Intracellular levels of adenosine 5'-triphosphate also dropped. These phenotypes are similar to those observed after phage superinfection, leading us to propose that at least some of the exclusion caused by the Rex proteins could be due to a change in their ratio following superinfection.
The bacteriophage T4 rII genes and the lambda rex (r exclusion) genes interact; rII mutants are unable to productively infect rex+ lambda lysogens. The relationship between rex and rII has been found to be quantitative, and plasmid clones of rex have excluded not only rII mutants but T4 wild type and most other bacteriophages as well. Mutations in the T4 motA gene substantially reversed exclusion of T4 by rex.
Complementation tests among previously isolated rex- mutants of bacteriophage lambda reveal that the mutants comprise two complementation groups, designated rexA and rexB. Because rexB- mutants complement prm- mutants, but rexA- mutants do not, it appears that the rexA gene is coordinately controlled with the cI (repressor) gene under the direction of PRM promoter, but that some other promoter is capable of directing the expression of rexB.
The coordinate expression from induced lambda prophages of pLit-rexB-tImm (late immunity transcription, LIT RNA) and po-oop-t(o) (OOP RNA) has remained unexplained. The initial assigned sequence for pLit bore no relationship to po. We have identified two promoter sites for independent rexB transcription, denoted here pLit2 and pLit1, which are separated by about 330 bp. The upstream pLit1 site shares with po a common 9 bp sequence between the -10 and -35 regions, with strong homology to aspects of the SOS box or LexA operator site. This sequence is also found within OOP RNA, suggesting that OOP RNA, or another regulatory factor recognizing the common sequence, was involved in the regulation of rexB expression and hence Rex exclusion. We measured the influence of OOP synthesis from plasmids on the Rex phenotype, finding that plasmids producing OOP can suppress Rex exclusion by a lambda prophage. The possibility was suggested that low level constitutive rexB transcription occurs from pLit2. Potential binding sites were identified for DnaA, for the LexA, CI and Cro repressors and for lambda O protein in the 80 nt DNA interval upstream from and including pLit1, suggesting a complex regulatory pattern for rexB expression from this promoter.
The rex genes of bacteriophage lambda were found to protect lysogenic Escherichia coliK host cells against killing by phage T4 rII, when compared in parallel to isogenic Rex(-) lysogens and nonlysogens. This protective effect was abrogated upon mutation of the host stationary-phase sigma factor RpoS. Rex(+) lysogens infected by T4 rII contracted, formed aggregates and shed flagella, thus resembling cells entering stationary phase. These phenotypes were accentuated in nonlysogenic cells carrying multicopy plasmids expressing rexA-rexB: cells were about two-fold contracted in length, expressed membrane-bound and detached flagella, were insensitive to infection by a variety of phages and clumped extensively; in addition, cultures of these cells were odorous. Our observations support the hypothesis that the Rex system can cause a stationary-phase-like response that protects the host against infection by T4 rII.
This paper describes a functionally related region in the genetic material of a bacteriophage that is finely subdivisible by mutation and by genetic recombination. The group of mutants resembles similar cases which have been observed in many organisms, usually designated as "pseudo-alleles." (See reviews by Lewis [1] and Pontecorvo [2].) Such cases are of special interest for their bearing on the structure and function of genetic determinants.
The phenomenon of genetic recombination provides a powerful tool for separating mutations and discerning their positions along a chromosome. When it comes to very closely neighboring mutations, a difficulty arises, since the closer two mutations lie to one another, the smaller is the probability that recombination between them will occur. Therefore, failure to observe recombinant types among a finite number of progeny ordinarily does not justify the conclusion that the two mutations are inseparable but can only place an upper limit on the linkage distance between them. A high degree of resolution requires the examination of very many progeny. This can best be achieved if there is available a selective feature for the detection of small proportions of recombinants. Some preliminary results are here presented of a program designed to extend genetic studies to the molecular (nucleotide) level.
Derivations and genotypes of some mutant derivatives of Escherichia coli K-12. In Escherichia coli and Sal-monella typhimurium: cellular and molecular biology
- B J Bachmann
Bachmann, B.J. 1987. Derivations and genotypes of some mutant derivatives of Escherichia coli K-12. In Escherichia coli and Sal-monella typhimurium: cellular and molecular biology. Vol. 2. Edited by F.C. Neidhardt, J.I. Ingraham, K.B. Low, B. Magasanik, M. Schaechter, and H.E. Umbarger. American Society for Micro-biology, Washington, D.C. pp. 1192–1219.