Journal of Bacteriology, August 2004, p . 5533-5537, Vol . 186, No . 16

Evidence of bar Minigene Expression and tRNA₂^IleSequestration as Peptidyl-tRNA₂^Ileduring Lambda Bacteriophage Development

Norma Angélica Oviedo de Anda, Luis Kameyama, José Manuel Galindo, Gabriel Guarneros, and Javier Hernandez-Sanchez^*

Departamento de Genética y Biología Molecular, CINVESTAV-IPN, San Pedro Zacatenco, México D.F . 07360, Mexico

Received 3 February 2004/ Accepted 12 May 2004

 
Lambda bacteriophage development is impaired in Escherichia coli cells defective for peptidyl (pep)-tRNA hydrolase (Pth) . Single-base-pair mutations (bar^–) that affect translatable two-codon open reading frames named bar minigenes (barI or barII) in the lambda phage genome promote the development of this phage in Pth-defective cells (rap cells) . When the barI minigene is cloned and overexpressed from a plasmid, it inhibits protein synthesis and cell growth in rap cells by sequestering as . Either or Pth may reverse these effects . In this paper we present evidence that both barI and barII minigenes are translatable elements that sequester as . In addition, overexpression of the barI minigene impairs the development even of bar^– phages in rap cells . Interestingly, tRNA or Pth may reestablish lambda phage development . These results suggest that lambda bar minigenes are expressed and is sequestered as during lambda phage development .

 
Bacteriophage lambda is unable to grow vegetatively in Escherichia coli (rap) mutants defective in peptidyl (pep)-tRNA hydrolase (Pth) activity (8, 9, 11) . Phage mutants that grow readily in the defective bacteria are affected in regions of the lambda genome named bar . One of these, barI, is located at the phage attachment site, attP, and another one, barII, is located within the ssb gene (9) . Overexpression of bar regions in plasmid vectors causes growth inhibition of Pth-defective E . coli cells (8, 10, 20) . Analogous constructs carrying lambda mutant bar regions are nontoxic (10, 26) . The nearly identical barI and barII sequences harbor minigenes, which are DNA segments whose transcripts contain a Shine-Dalgarno sequence appropriately spaced for translation from either AUG AUA UAA (barI) or AUG AUA UGA (barII) sequences . Overexpression of bar minigenes under limiting Pth activity in vivo and in vitro leads to the accumulation of (), and purified preparations of Pth protein or are able to reverse minigene-mediated inhibition of protein synthesis in vitro (12, 25) .

Minigene-mediated cell toxicity achieved in Pth-defective cells by the use of multicopy plasmids differs from lambda phage exclusion, in which both barI and barII minigenes must be transcribed to reduce phage development (9) . Both the barI and barII regions are parts of the lambda left operon and are transcribed by the protein RNA polymerase antitermination complexes which are initiated at p_L (3, 23) . Thus, the mutations sex1 of lambda, which results in a phage defective in the p_L promoter (21), and nutL44, which prevents transcript elongation beyond the transcription terminator t_L1 (22), develop successfully in Pth-defective cells . On the contrary, those mutations that enhance the expression of bar regions inhibit lambda phage development . The mutations intC266, which causes constitutive transcription from the p_I promoter across the attP site (24), and cro27, which results in a phage defective in the repression of p_L (7), make these phages unable to develop in Pth-defective cells . Therefore, the stringency of lambda phage exclusion in Pth-defective cells depends on the degree of transcription through the barI and barII regions (9) .

The data presented in the present paper indirectly suggest that the bar minigene regions are expressed during lambda phage development . Additionally, the capacity of Pth or to promote lambda phage development in Pth-defective cells indicates that is probably sequestered as . We argue about the role of these observed conditions as part of a potentially interesting interaction between the phage and the host, which could be involved in a type of mini-open reading frame (ORF)-mediated translational regulation of gene expression .

or Pth may alleviate lambda barI and barII minigene-mediated cell growth inhibition in Pth-defective cells. Expression of lambda bacteriophage barI and barII minigenes from plasmid constructs inhibits protein synthesis and cell growth in Pth-defective cells (26) . In addition, the barI minigene expressed in vitro accumulates under limiting Pth activity (12). supplementation of Pth-defective cells reverses barI-mediated cell growth inhibition . Thus, we extended these investigations to the lambda barII minigene to ascertain whether the behavior of barII parallels that of barI .

E . coli C600 cI857 or Pth-defective C600 cI857 (rap) cells were transformed with any of the following plasmids containing wild-type or mutant minigenes under the p_L promoter (17, 26): pFGbarI (bearing the wild-type barI minigene), bar101 (with an AUG to AUA substitution at the first codon of the barI mini-ORF), pCMbarII (containing the barII minigene), and pCMbar205 (with a base pair substitution from AUA to AUG at the second codon of the barII mini-ORF) . For Pth supplementation, cells were transformed with pGREC (harboring the E . coli pth gene) (G . Rosas-Sandoval, unpublished results), and for /supplementation, cells were transformed with plasmids pDC952 and pI289, which were derived from pACYC184 (2) by cloning the and genes, respectively (4) . For clarity, plasmids pGREC, pDC952, and pI289 are designated in this paper as pPth, pArg4, and pIle2, respectively . Transcription through the bar minigene was derepressed at 43°C via a thermosensitive lambda cI repressor in a cryptic prophage in C600 rap cells (see reference 26) . The effect of Pth or on the viability of C600 cI857 rap cells transformed with either barI or barII minigene-containing plasmids was monitored for 120 min . The results (Fig . 1A) revealed that Pth reversed barII minigene-mediated cell growth inhibition, as has previously been reported for barI (26) . Under the conditions tested, had a moderate but significant effect on cell growth restoration (Fig. 1B) . pArg4 containing the gene or pACYC184 where these tRNA genes were cloned had no effect (data not shown) . These results suggest that both barI and barII minigenes sequester as . Therefore, both minigenes are translatable entities when they are cloned and expressed outside of their context .

 
Translation of minigenes that sequester as reduces the development of mutant lambda bacteriophages in Pth-defective cells. Bacteriophage lambda is unable to grow in E . coli mutants defective in Pth activity (8, 9, 11) . Phage mutants in which the translatability of barI or barII minigenes is impaired increase their capacity to grow in Pth-defective cells (9) . These antecedents, together with the above observations, suggest that bar minigenes are expressed and that the activity of Pth is required during lambda phage development . To further analyze the role of these elements in lambda development, we artificially exacerbated their effect by a controlled expression of the barI minigene from a multicopy plasmid in wild-type or rap cells under conditions where cell growth is not apparently affected .

Lysates of lac trp W205 red114 imm434, bar101, and bar205 bacteriophages, which are derivatives able to grow at 32°C, were prepared (9, 10) . Phage dilutions from lysates containing the same titer (10 µl) were spotted on cell lawns prepared by pouring 2.5 ml of soft tryptone broth with 100 µl of bar minigene-expressing C600 or Pth-defective C600 cells over a Luria-Bertani (LB) medium plate .

The lambda bar101 or bar205 mutants that were affected in only one of the minigenes were able to grow in Pth-defective cells, although not as efficiently as in wild-type cells (Fig . 2B) . This may be due to the fact that they do not demand as much as wild-type lambda . Accordingly, the development of lambda bar101 or bar205 mutants was dramatically reduced when the limited levels were further exhausted by expressing the -sequestering barI minigene (Fig . 2D) . Importantly, the development of both mutant and wild-type phages is also inhibited in Pth-defective cells under conditions where cell growth is not affected (Fig . 2C) . These results indicate that the degree of lambda phage development depends on the Pth cell activity and cell levels and on the translatability of bar minigenes .

 
Pth or restores lambda bacteriophage development in Pth-defective cells. The reduced lambda phage development in Pth-defective cells suggests that bar minigenes may produce which may not readily be hydrolyzed by the low Pth activity levels . Therefore, Pth supplementation of Pth-defective cells should restore the cells' capacity to support lambda phage development . Phage development in Pth-defective cells supplemented with Pth was comparable to that in wild-type cells (Fig . 2A and 3A) . If barI-mediated reduction of phage development in Pth-defective cells were caused by starvation of free sequestered as , supplementing the cells with should also reestablish lambda phage development . As expected, wild-type phage development in Pth-defective cells was further enhanced by supplementing (Fig. 3B) . pArg4 containing the gene or pACYC184 in which Pth or the tRNA genes were cloned had no effect (data not shown) . Optimal phage development was promoted by supplementation, presumably because the size of the cell pool is increased .

 
The results presented in this paper suggest that bar regions are translated during lambda phage development . In addition, the promoting activities of Pth and in lambda phage development strongly indicate that is sequestered as .

Indirect evidence of the translatability of the bar regions stems from previous work and the results presented in Fig . 1A, in which experiments the barI and barII regions are overexpressed by the use of multicopy plasmid constructs . Under these circumstances, minigenes become toxic in Pth-defective cells . Even though the barI and barII regions cloned in the constructions used in this work differ broadly in their nucleotide sequences, except for the ORF and a 6-bp tract beyond the termination codons, they show the same properties of cell growth inhibition and growth restoration by Pth or .

Toxicity (cell growth and protein synthesis inhibition) in Pth-defective cells is the result of tRNA sequestration as pep-tRNA during bar minigene overexpression from multicopy plasmids . This situation differs from the exclusion of lambda phage where both barI and barII presumably must be transcribed and translated to block phage development in Pth-defective cells . In addition, our data indicate that is also sequestered as . However, we have been unable to detect in total cell extracts by a Northern blot assay (16) because the concentration of and presumably the corresponding levels produced in the cell are very low (6, 13) . In this way, lambda phage development may be impaired in Pth-defective cells, because the scarce is promptly sequestered as , and this in turn is not readily hydrolyzed by the limiting Pth activity .

The pep-tRNA accumulated may provoke protein synthesis inhibition per se (1) and/or deplete the levels of tRNA under a critical concentration incompatible with phage development and/or cell protein synthesis . Since an excess of specific tRNA in vitro (12) or in vivo (25) suppressed protein synthesis inhibition (12) and restored phage development in Pth-defective cells (Fig . 3B), the latter inference is more plausible .

We infer that minigene expression and production should occur during normal phage development in wild-type cells . However, as soon as is produced, it is hydrolyzed by normal Pth activity, and phage development is not impaired . It is feasible to attain high pep-tRNA levels in wild-type cells by expressing barI from a pUC-based vector (J . G . Valadez, unpublished results) . This plasmid occurs in about 10-fold more copies per cell than the pBR322-based vector (15) used to do the experiments in this work . An uncontrolled overexpression from this derivative is lethal even in wild-type cells . In addition, barI overexpression using wild-type cell extracts also inhibits protein synthesis, albeit less stringently than with Pth-defective cell extracts (12) . However, in these cases an exaggerated overexpression of a minigene or even a gene may compete with other genes for the translational machinery, leading to an unspecific inhibition of protein synthesis irrelevant for the -sequestering mechanism proposed in the present work (14) . Thus, the amount of produced by wild-type lambda phage in wild-type cells or by mutant barI or barII phage in Pth-defective cells should not overcome the capacity of Pth activity to hydrolyze it . Accordingly, when the pool was artificially reduced by overexpressing the -sequestering barI minigene, the development of mutant barI or barII phage was also impaired in Pth-defective cells (Fig . 2D) . These results are the basis of the argument that minigene-mediated toxicity or phage exclusion in Pth-defective cells depends on both the level of Pth activity and minigene expression . The suggestion that these phenomena are related to the -sequestering mechanism and the expression of AUA-containing bar minigenes is also supported, at least in the plasmid system, by the fact that the change of the rare AUA to the common synonymous AUU codon renders the barI minigene nontoxic in Pth-defective cells (18; R . Cruz-Vera, unpublished results) .

Experiments performed in vitro have shown different parameters affecting minigene toxicity, including the nature of the translational signals (Shine-Dalgarno sequence, initiation codon, stop codon, and last sense codon), pep-tRNA drop-off, pep-tRNA hydrolysis rate by Pth, and minigene recycling (12, 19, 25) . Minigenes contain the necessary signals for translation; however, their toxicity is always associated with an inefficient translational termination and pep-tRNA release from the ribosome . This could be due in part to ribosome pausing at the rare AUA codon and to the proximity of the initiation and stop codons (5, 12) .

A computer program designed to recognize potentially translatable short ORFs in prokaryote genomes identified 118 possible minigenes in lambda DNA . However, bar-like minigenes (toxic in Pth-defective bacteria) represented only 10% of the identified clones (18) . Among these minigenes, barI and barII contribute greatly to the reduced lambda phage developing capacity in Pth-defective cells, as the mutations that affect the translatability of any of these minigenes indicate . However, their role in lambda biology is yet to be determined . Lambda phage might have evolved the bar minigene system for a fine translational downregulation of lambda genes containing the rare ATA codon . In fact, a computer analysis shows a high frequency of ATA-containing genes in the regulatory region of the lambda genome (F . de la Vega, unpublished results) . Alternatively, this system might also be a general mechanism to inhibit host translation of ATA-containing genes, since the translation of bar minigenes poses an unusual demand on the cellular pool of .

 
This work was supported by CONACyT grant 34836-N (to J.H.-S.), CONACyT grant 28401N, and COSNET grant 1400.99.P (to G.G.) . N.A.O.D.A . was supported by fellowships from CONACyT and COSNET .

We thank M . A . Magos Castro for technical assistance .

 
* Corresponding author . Mailing address: Departamento de Genética y Biología Molecular, CINVESTAV-IPN, Av . Instituto Politécnico Nacional 2508, San Pedro Zacatenco, México D.F . 07360, Mexico . Phone: 52-55 5061 3800, ext . 5340 . Fax: 52-55 5747 7100 . E-mail: javierh@mail.cinvestav.mx.

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