Evidence that there are only two tRNA(Phe) genes in Escherichia coli.

pheV, one of the genes that code for tRNA(Phe), was deleted from the chromosome of a strain of Escherichia coli K-12. As a consequence of this mutation, expression of pheA, the gene for chorismate mutase P-prephenate dehydratase, the first enzyme in the terminal pathway of phenylalanine biosynthesis, was derepressed. Similar derepression of pheA has been reported in pheR mutants of E. coli K-12 (J. Gowrishankar and J. Pittard, J. Bacteriol. 150:1130-1137, 1982). Attempts to introduce a pheR mutation into the delta pheV strain failed under circumstances suggesting that this combination of mutations is lethal. Southern blot analysis of pheV+ and delta pheV strains indicated that there are only two tRNA(Phe) genes in E. coli. It is recommended that the names pheU and pheV be retained for these genes.     

Mutants of pheV in Escherichia coli affecting control by attenuation of the pheS, T and pheA operons. Two distinct mechanisms for de-attenuation

Two mutants of pheV, a gene coding for tRNA(Phe) in Escherichia coli, were previously isolated because they affect attenuator control of the pheS, T operon when the mutant pheV genes are carried by the plasmid pBR322. We show that the two mutants (A44 and A46) affect attenuator control by different mechanisms. The effect of mutant A44 on pheS, T expression can be progressively decreased by overproduction of Phe-tRNA synthetase, consistent with the mutant tRNA acting as a competitive inhibitor of the enzyme. By contrast, the effect on attenuation of mutant A46 increases with overproduction of Phe-tRNA synthetase, indicating that the mutant must be charged to affect attenuation; we propose that this mutant affects translation directly and causes derepression by competing with wild-type tRNA in translation of the attenuator region leader peptide. Mutant A46 but not mutant A44 leads to further de-attenuation in a miaA background. The presence of two different mechanisms for de-attenuation is further indicated by the finding that a second attenuator controlled by Phe codon translation, from the pheA operon, is affected quite differently by the mutant tRNAs. Finally, experiments involving the introduction of the mutations A44 and A46 into an amber suppressor derived from tRNA(Phe) suggest that both species can function in protein synthesis but with reduced efficiency; mutant A46 is less efficient than mutant A44, consistent with a defect in elongation.     

Mutants affecting tRNA(Phe) from Escherichia coli. Studies of the suppression of thermosensitive phenylalanyl-tRNA synthetase

Four mutants of pheV, a gene coding for tRNA(Phe) in Escherichia coli, share the characteristic that when carried in the plasmid pBR322, they lose the capacity of wild-type pheV to complement the thermosensitive defect in a mutant of phenylalanyl-tRNA synthetase. One of these mutants, leading to the change C2----U2 in tRNA(Phe), is expressed about 10-fold lower in transformed cells than wild-type pheV. This mutant, unlike the remaining three (G15----A15, G44----A44, m7G46----A46), can recover the capacity to complement thermosensitivity when carried in a plasmid of higher copy number. The other three mutants, even when expressed at a similar level, remain unable to complement thermosensitivity. A study of charging kinetics suggests that the loss of complementation associated with these mutants is due to an altered interaction with phenylalanyl-tRNA synthetase. The mutant gene pheV (U2), when carried in pBR322, can also recover the capacity to complement thermosensitivity through a second-site mutation outside the tRNA structural gene, in the discriminator region. This mutation, C(-6)----T(-6), restores expression of the mutant U2 to about the level of wild-type tRNA(Phe).     

Molecular cloning of pheR in Escherichia coli K-12.

The regulator gene pheR, which in Escherichia coli controls the expression of pheA, the structural gene for chorismate mutase P-prephenate dehydratase, was cloned on to multicopy plasmids directly from the E. coli chromosome; this was achieved with the aid of the tetracycline resistance transposon, Tn10, that had been inserted very close to the pheR gene. Subsequently, pheR was subcloned on a 1.1-kilobase-pair fragment on the plasmid vector pBR322; its position on the plasmid was localized by the method of gamma delta-mediated transpositional inactivation. The pheR gene product was identified in maxicells and found to be a protein of subunit molecular weight 19,000, suggesting that the coding segment of the gene is about 500 nucleotide pairs long.     

1H-15N NMR studies of Escherichia coli tRNA(Phe) from hisT mutants: a structural role for pseudouridine

Escherichia coli tRNA(Phe)U39 was isolated from a specially constructed bacterial strain (DD1003/pRK3) carrying mutations in the hisT gene (the structural gene for tRNA pseudouridine synthase I) and in the pyrB gene (uracil auxotrophy). The pheU gene for tRNA(Phe) under control of the native tRNA promoter was on a multicopy plasmid and gave up to 40-fold overproduction of tRNA(Phe)U39. The double mutant permitted efficient incorporation of [3-15N]uracil, resulting in greater than 95% 15N enrichment of uracil-derived bases. 1H and 1H-15N NMR experiments were used to assign the low-field proton resonances to specific hydrogen-bonding interactions. 1H NMR assignments indicate that tRNA(Phe)U39 has a structure similar to that of native tRNA(Phe) except in the anticodon region where replacement of pseudouridine (psi) at position 39 with uridine (U) destabilizes hydrogen-bonding interactions at the base of the anticodon stem. We propose that U----psi modifications further stabilize interactions normally available to U by providing an additional locus for hydrogen bonding to the pyrimidine ring.     

Characterization and evidence of mobilization of the LEE pathogenicity island of rabbit-specific strains of enteropathogenic Escherichia coli

We have characterized the LEE pathogenicity islands (PAIs) of two rabbit-specific strains of enteropathogenic E. coli (REPEC), 83/39 (serotype O15:H-) and 84/110-1 (O103:H2), and have compared them to homologous loci from the human enteropathogenic and enterohaemorrhagic E. coli strains, E2348/69 and EDL933, and another REPEC strain, RDEC-1. All five PAIs contain a 34 kb core region that is highly conserved in gene order and nucleotide sequence. However, the LEE of 83/39 is significantly larger (59 540 basepairs) than those of the human strains, which are less than 44 kb, and has inserted into pheU tRNA. The regions flanking the 34 kb core of 83/39 contain homologues of two putative virulence determinants, efa1/lifA and senA. The LEE of 84/110-1 is approximately 85 kb and is located at pheV tRNA. Its core is almost identical to those of 83/39 and RDEC-1, apart from a larger espF gene, but its flanking regions contain trcA, a putative virulence determinant of EPEC. All three REPEC LEE PAIs contain a gene for an integrase, Int-phe. The LEE PAI of 84/110-1 is also flanked by short direct repeats (representing the 3'-end of pheV tRNA), suggesting that it may be unstable. To investigate this possibility, we constructed a LEE::sacB derivative of 84/110-1 and showed that the PAI was capable of spontaneous deletion. We also showed that Int-phe can mediate site-specific integration of foreign DNA at the pheU tRNA locus of E. coli DH1. Together these results indicate possible mechanisms of mobilization and integration of the LEE PAI.     

Regulatory gene of phenylalanine biosynthesis (pheR) in Salmonella typhimurium

4-Fluorophenylalanine-resistant mutants of Salmonella typhimurium were isolated in which synthesis of chorismate mutase P-prephenate dehydratase (specified by pheA) was highly elevated. Transduction analysis showed that the mutation affecting pheA activity was not linked to pheA, and conjugation and merodiploid analysis indicated that it was in the 95- to 100-min region of the Salmonella chromosome. Evidence is presented for the hypothesis that the mutation responsible for constitutivity of chorismate mutase P-prephenate dehydratase occurred in pheR, a gene specifying a cytoplasmic product that affected pheA. pheR mutants were found to carry a second mutation, tyrO. The tyrO mutation acts cis to cause increased levels of the tyrosine biosynthetic enzymes 3-deoxy-d-arabinoheptulosonate 7-phosphate synthetase (tyr) and prephenate dehydrogenase, but it has no effect on regulation of pheA.     

CTnscr94, a conjugative transposon found in enterobacteria.

Conjugational transposons are important for horizontal gene transfer in gram-positive and gram-negative bacteria, but have not been reported yet for enteric bacteria. Salmonella senftenberg 5494-57 has previously been shown to transfer by conjugation genes for a sucrose fermentation pathway which were located on a DNA element called scr-94. We report here that the corresponding scr genes for a phosphoenolpyruvate-dependent sucrose:phosphotransferase system and a sucrose metabolic pathway are located on a large (ca. 100 kb) conjugative transposon renamed CTnscr94. The self-transmissible element integrates at two specific attachment sites in a RecA-independent way into the chromosome of Escherichia coli K-12 strains. One site was identified within pheV, the structural gene for a tRNA(Phe). Sequencing of both ends of CTnscr94 revealed the presence of the 3' part of pheV on one end such that after integration of the element, a complete pheV gene is retained. CTnscr94 represents, to our knowledge, the first conjugational transposon found in enteric bacteria.     

Self-transmissibility of the integrative and conjugative element ICEPm1 between clinical isolates requires a functional integrase, relaxase, and type IV secretion system

Integrative and conjugative elements (ICEs), which are chromosomal mobile elements, can conjugatively transfer between bacteria. Recently, we identified a genomic island of Proteus mirabilis, a common agent of catheter-associated urinary tract infection (UTI), that possesses all the properties consistent with an ICE. This element, designated ICEPm1, is highly conserved in other causative agents of UTI, suggesting its mobility. We demonstrate that ICEPm1 can actively excise from the chromosome in a clonal population of bacteria and that this excision is integrase dependent. Although in P. mirabilis HI4320, ICEPm1 is annotated as integrated into the phenylalanine tRNA gene pheV, we show that ICEPm1 can integrate into either pheV or pheU. We determined that ICEPm1 transfers at a frequency of 1.35 × 10(-5) transconjugants/donor to ICEPm1-deficient P. mirabilis using plate mating assays with clinical isolates. Insertional inactivation of a putative integrase gene on ICEPm1 decreased transfer frequencies of ICEPm1 to below the limit of detection. Mutation of the relaxase of ICEPm1 also eliminates transfer and demonstrates that this element is indeed self-transmissible and not transferred in trans, as are some mobilizable genomic islands. Together, these findings clearly demonstrate that ICEPm1 can actively excise from the chromosome in an integrase-dependent manner, dynamically integrate into both phenylalanine tRNA genes, and transfer into clinical strains using its own conjugation machinery.     

Localization of the insertion site and pathotype determination of the locus of enterocyte effacement of shiga toxin-producing Escherichia coli strains

Of 220 Shiga toxin-producing Escherichia coli (STEC) strains collected in central France from healthy cattle, food samples, and asymptomatic children, 12 possessed the eae gene included in the locus of enterocyte effacement (LEE) pathogenicity island. Based on gene typing, we observed 7 different eae espA espB tir pathotypes among the 12 STEC strains and described the new espAbetav variant. As previously observed, the O157 serogroup is associated with eaegamma, O26 is associated with eaebeta, and O103 is associated with eaeepsilon. However, the unexpected eaezeta allele was detected in 5 of the 12 isolates. PCR amplification and pulsed-field gel electrophoresis using the I-CeuI endonuclease followed by Southern hybridization indicated that the LEE was inserted in the vicinity of the selC (three isolates), pheU (two isolates), or pheV (six isolates) tRNA gene. Six isolates harbored two or three of these tRNA loci altered by the insertion of integrase genes (CP4-int and/or int-phe), suggesting the insertion of additional foreign DNA fragments at these sites. In spite of great genetic diversity of LEE pathotypes and LEE insertion sites, bovine strains harbor alleles of LEE genes that are frequently found in clinical STEC strains isolated from outbreaks and sporadic cases around the world, underscoring the potential risk of the bovine strains on human health.     

Defective regulation of the phenylalanine biosynthetic operon in mutants of the phenylalanyl-tRNA synthetase operon.

Among mutants of Escherichia coli resistant to p-fluorophenylalanine (PFP) were some with constitutive expression of the phenylalanine biosynthetic operon (the pheA operon). This operon is repressed in the wild type by phenylalanine. The mutation in three of these mutants mapped in the aroH-aroD region of the E. coli chromosome at 37 min. A plasmid bearing wild-type DNA from this region restored p-fluorophenylalanine sensitivity and wild-type repression of the pheA operon. Analysis of subclones of this plasmid and comparison of its restriction map with published maps indicated that the mutations affecting regulation of the pheA operon lie in the structural genes for phenylalanyl-tRNA synthetase, pheST, probably in pheS. Thus, the pheST operon has a role in the regulation of phenylalanine biosynthesis, the most likely being that wild-type phenylalanyl-tRNA synthetase maintains a sufficient intracellular concentration of Phe-tRNA(Phe) for attenuation of the pheA operon in the presence of phenylalanine. A revised gene order for the 37-min region of the chromosome is reported. Read clockwise, the order is aroD, aroH, pheT, and pheS.     

Identification of clones carrying an E. coli tRNAPhe gene by suppression of phenylalanyl-tRNA synthetase thermosensitive mutants

Two libraries of cloned E. coli DNA were screened for plasmids which complemented thermosensitive phenylalanyl-tRNA synthetase mutants. Four plasmids were isolated which complemented pheS and pheT thermosensitive mutations but which do not carry pheS or pheT, the structural genes for phenylalanyl-tRNA synthetase. All these plasmids increased the intracellular tRNAPhe concentration. Three plasmids were shown to carry the structural gene for tRNAPhe which we call pheU. By restriction enzyme analysis, DNA blotting and DNA:tRNA hybridization, pheU was localised to a 280 bp fragment within a 5.6 kb PstI restriction fragment of E.coli DNA.     

Regulation of tyrosine and phenylalanine biosynthesis in Salmonella.

Several types of 4-fluorophenylalanine resistant mutants were isolated. In one type of mutant DAHP synthetase (tyr) and prephenate dehydrogenase were coordinately derepressed. The mutation was linked to aroF and tyrA and was cis- dominant by merodiploid analysis, thus confirming that it is an operator constitutive mutation (tyrOc). A second type of mutation showed highly elevated levels of tyrosine pathway enzymes which were not repressed by L-tyrosine. It was unlinked to tyrA and aroF, and was trans-recessive in merodiploids. These properties were attributed to a mutation in a regulator gene, tyrR (linked to pyr F), that resulted in altered or non-functional aporepressor. Hence tyrO, tyrA, and aroF constitute an operon regulated by tyrR. In a third type of mutation chorismate mutase P-prephenate dehydratase was highly elevated. It was not linked to pheA, was located in the 95--100 min region of the Salmonella chromosome, and was recessive to the wild type gene in merodiploids. A mutation was, therefore, indicated in a regulatory gene, pheR, which specified an aporepressor for regulating pheA. DAHP synthetase (phe), specified by aroG, was not regulated by pheR, but was derepressed in one of the tyrR mutants, suggesting that as in Escherichia coli tyrR may regulate DAHP synthetase(phe) and DAHP synthetase (tyr) with the same aporepressor. A novel mutation in chorismate mutase is described.     

The nucleotide sequences of two tRNAAsn genes from tobacco chloroplasts

Recombinant plasmids which contain EcoRI fragments of tobacco chloroplast DNA carrying tRNA genes were constructed. Plasmids pTC211 and pTC293 contain the base sequences for tRNAAsn in their 1.4 and 1.1 Md EcoRI fragments, respectively. These two tRNA sequences are identical and are; 5'-TCCTCAGTAGCTCAGTGGTAGAGCGGTCGGCTGTTAACCGATTGGTCGTAGGTTCGAATCCTACTTGGGGAG-3'. Each tRNAAsn gene is located at about 0.9 kb apart from the distal end of each 5S rRNA gene and is coded for by the DNA strand opposite from that of the rRNA genes.     

Suppression of a double missense mutation by a mutant tRNA(Phe) in Escherichia coli.

We report here the isolation of a mutant tRNAPhe that suppresses a double missense auxotrophic mutation in trpA of Escherichia coli, trpA218. The doubly mutant protein product differs from wild-type TrpA by the replacements of Phe22 by Leu and Gly211 by Ser. A partial revertant TrpA phenotype can be obtained from trpA218 by changing either Leu22 back to Phe or Ser211 back to Gly. Translational suppressors were previously obtained that act at codon 211, replacing the Ser211 in the TrpA218 protein, presumably with Gly. In the present study, we selected for trpA218 suppressors caused by mutation of a cloned tRNAPhe gene, pheV. DNA sequence analysis of the suppressor isolated reveals a singular structural alteration, changing the anticodon from 5'-GAA-3' to 5'-GAG-3'. Sequencing of trpA218 confirmed the likely identity of Leu22 as CUC. The new missense suppressor, designated pheV(SuCUC), is lethal to the cell when highly expressed, as from a high copy number plasmid. This may be due to efficient replacement of Leu by Phe at CUC (and, probably, CUU) codons throughout the genome. We anticipate that pheV(SuCUC) will prove, like other missense suppressors, to be extremely useful in studies on the specificity and accuracy of decoding.