Queuosine modification in tRNA and expression of the nitrate reductase in Escherichia coli.

In eubacteria the modified nucleoside queuosine is present in tRNAAsn, tRNAAsp, tRNAHis and tRNATyr. A precursor of queuine, pre-queuine, is synthesized from GTP, inserted into the first position of the anticodon of the corresponding tRNAs by a specific tRNA-guanine transglycosylase and further modified to queuosine. Isogenic pairs of Escherichia coli, containing or lacking the tRNA-transglycosylase (JE 7335, tgt+ lacZ+ and JE 7337, tgt- lacZ+; JE 7334, tgt+ lacZ- and JE 7336, tgt- lacZ-), have been employed to study the function of queuosine in tRNA. Compared with the tgt+ strain (JE 7335), the tgt- mutant (JE 7337) grown under anaerobic conditions, is defective with respect to the nitrate respiration system, in which electrons are transported from D(-)-lactate via quinone and cytochrome bNO3-(556) to nitrate. Low temperature cytochrome spectra of the anaerobically grown tgt- mutant show a lowered amount of type b cytochromes involving the spectrum of cytochrome bNO3-(556). In the case of the anaerobically grown tgt- mutant three proteins are missing in the protein pattern of cytoplasmic membranes. Their mol. wts. correspond to those of the subunits of the nitrate reductase complex. In contrast to the tgt+ strains (JE 7334, JE 7335) both tgt- mutants (JE 7336, JE 7337) cannot grow on lactate under anaerobic conditions with nitrate offered as electron acceptor and NO3- is not reduced to NO2-. A possible link between Q-modification of tRNAs, the synthesis of proteins of the nitrate reductase complex and the synthesis of menaquinone or ubiquinone is discussed.     

Cytochrome o (cyoABCDE) and d (cydAB) oxidase gene expression in Escherichia coli is regulated by oxygen, pH, and the fnr gene product.

The aerobic respiratory chain of Escherichia coli contains two terminal oxidases that catalyze the oxidation of ubiquinol-8 and the reduction of oxygen to water. They are the cytochrome o oxidase complex encoded by cyoABCDE and the cytochrome d oxidase complex encoded by cydAB. To determine how these genes are regulated in response to a variety of environmental stimuli, including oxygen, we examined their expression by using lacZ protein fusions in wild-type and fnr mutant strains of E. coli. Anaerobic growth resulted in a 140-fold repression of cyoA'-'lacZ expression relative to aerobic growth and a 3-fold increase in cydA'-'lacZ expression. Anaerobic repression of both fusions was mediated in part by the fnr gene product, as evidenced by a 30-fold derepression of cyoA'-'lacZ expression and a 4-fold derepression of cydA'-'lacZ expression in an fnr deletion strain. Supplying wild-type fnr in trans restored wild-type repression for both fusions. Fnr thus functions as an anaerobic repressor of both cyoABCDE and cydAB expression. Reduced-minus-oxidized difference spectrum analyses of cell membranes confirmed the effect of the fnr gene product on the production of cytochrome d oxidase in the cell. Based on the pattern of anaerobic cydAB expression observed, we propose the existence of a second, as yet unidentified, regulatory element that must function either to activate cydAB expression as oxygen becomes limiting or to repress cydAB expression aerobically. Whereas cytochrome o oxidase encoded by cyoABCDE appears to be produced only under oxygen-rich growth conditions, in keeping with its biochemical properties, cytochrome d oxidase is expressed moderately aerobically and is elevated yet further when oxygen becomes limiting so that the organism can cope better under oxygen starvation conditions. We also examined cyoABCDE and cydAB expression in response to growth on alternative carbon compounds and to changes in the culture medium pH and osmolarity.     

A single base change in the acceptor stem of tRNA(3Leu) confers resistance upon Escherichia coli to the calmodulin inhibitor, 48/80

We have isolated several classes of spontaneous mutants resistant to the calmodulin inhibitor 48/80 which inhibits cell division in Escherichia coli K12. Several mutants were also temperature sensitive for growth and this property was exploited to clone a DNA fragment from an E. coli gene library restoring growth at 42 degrees C and drug sensitivity at 30 degrees C in one such mutant. Physical and genetic mapping confirmed that both the mutation and the cloned DNA were located at 15.5 min on the E. coli chromosome at a locus designated feeB. By subcloning, complementation analysis and sequencing, the feeB locus was identified as identical to the tRNA(CUALEU) gene. When the mutant locus was isolated and sequenced, the mutation was confirmed as a single base change, C to A, at position 77 in the acceptor stem of this rare Leu tRNA. In other studies we obtained evidence that this mutant tRNA, recognizing the rare Leu codon, CUA, was defective in translation at both permissive and non-permissive temperatures. The feeB1 mutant is defective in division and shows a reduced growth rate at non-permissive temperature. We discuss the possibility that the mutant tRNA(3Leu) is limiting for the synthesis of a polypeptide(s), requiring several CUA codons for translation which in turn regulates in some way the level or activity of the drug target, a putative cell cycle protein.     

Mutation of the miaA gene of Agrobacterium tumefaciens results in reduced vir gene expression.

vir regulon expression in Agrobacterium tumefaciens involves both chromosome- and Ti-plasmid-encoded gene products. We have isolated and characterized a new chromosomal gene that when mutated results in a 2- to 10-fold reduction in the induced expression of vir genes by acetosyringone. This reduced expression occurs in AB minimal medium (pH 5.5) containing either sucrose or glucose and containing phosphate at high or low concentrations. The locus was cloned and used to complement A. tumefaciens strains harboring Tn5 insertions in the gene. Sequence analysis of this locus revealed an open reading frame with strong homology to the miaA locus of Escherichia coli and the mod5 locus of Saccharomyces cerevisiae. These genes encode tRNA: isopentenyltransferase enzymes responsible for the specific modification of the A-37 residue in UNN codon tRNA species. The function of the homologous gene in A. tumefaciens was proven by genetic complementation of E. coli miaA mutant strains. tRNA undermodification in A. tumefaciens miaA mutant strains may reduce vir gene expression by causing a reduced translation efficiency. A slight reduction in the virulence of these mutant Agrobacterium strains on red potato plants, but not on tobacco, tomato, kalanchoe, or sunflower plants, was observed.     

Nitric oxide formation by Escherichia coli. Dependence on nitrite reductase,the NO-sensing regulator Fnr,and flavohemoglobin Hmp

Nitric oxide (NO) is a key signaling and defense molecule in biological systems. The bactericidal effects of NO produced, for example, by macrophages are resisted by various bacterial NO-detoxifying enzymes, the best understood being the flavohemoglobins exemplified by Escherichia coli Hmp. However, many bacteria, including E. coli, are reported to produce NO by processes that are independent of denitrification in which NO is an obligatory intermediate. We demonstrate using an NO-specific electrode that E. coli cells, grown anaerobically with nitrate as terminal electron acceptor, generate significant NO on adding nitrite. The periplasmic cytochrome c nitrite reductase (Nrf) is shown, by comparing Nrf+ and Nrf- mutants, to be largely responsible for NO generation. Surprisingly, an hmp mutant did not accumulate more NO but, rather, failed to produce detectable NO. Anaerobic growth of the hmp mutant was not stimulated by nitrate, and the mutant failed to produce periplasmic cytochrome(s) c, leading to the hypothesis that accumulating NO in the absence of Hmp inactivates the global anaerobic regulator Fnr by reaction with the [4Fe-4S]2+ cluster (Cruz-Ramos, H., Crack, J., Wu, G., Hughes, M. N., Scott, C., Thomson, A. J., Green, J., and Poole, R. K. (2002) EMBO J. 21, 3235-3244). Fnr thus failed to up-regulate nitrite reductase. The model is supported by the inability of an fnr mutant to generate NO and by the restoration of NO accumulation to hmp mutants upon introducing a plasmid encoding Fnr* (D154A) known to confer activity in the presence of oxygen. A cytochrome bd-deficient mutant retained NO-generating activity. The present study reveals a critical balance between NO-generating and -detoxifying activities during anaerobic growth.     

Glutamine is incorporated at the nonsense codons UAG and UAA in a suppressor-free Escherichia coli strain

Readthrough of the nonsense codons UAG, UAA, and UGA is seen in Escherichia coli strains lacking tRNA suppressors. Earlier results indicate that UGA is miscoded by tRNA(Trp). It has also been shown that tRNA(Tyr) and tRNA(Gln) are involved in UAG and UAA decoding in several eukaryotic viruses as well as in yeast. Here we have investigated which amino acid(s) is inserted in response to the nonsense codons UAG and UAA in E. coli. To do this, the stop codon in question was introduced into the staphylococcal protein A gene. Protein A binds to IgG, which facilitates purification of the readthrough product. We have shown that the stop codons UAG and UAA direct insertion of glutamine, indicating that tRNA(Gln) can read the two codons. We have also confirmed that tryptophan is inserted in response to UGA, suggesting that it is read by tRNA(Trp).     

Ribosome-initiator tRNA complex as an intermediate in translation initiation in Escherichia coli revealed by use of mutant initiator tRNAs and specialized ribosomes.

For functional studies of mutant Escherichia coli initiator tRNAs in vivo, we previously described a strategy based on the use of tRNA genes carrying an anticodon sequence change from CAU to CUA along with a mutant chloramphenicol acetyltransferase (CAT) gene carrying an initiation codon change from AUG to UAG. Surprisingly, under conditions where the mutant initiator tRNA is optimally active, the CAT gene with the UAG initiation codon produced more CAT protein (3- to 9-fold more depending on the conditions) than the wild-type CAT gene. Here we show that two new mutant CAT genes having GUC and AUC initiation codons also produce more of the CAT protein in the presence of the corresponding mutant initiator tRNAs. These results are most easily understood if assembly of the 30S ribosome-initiator tRNA-mRNA initiation complex in vivo proceeds with the 30S ribosome binding first to the initiator tRNA and then to the mRNA. In cells overproducing the mutant initiator tRNAs, most ribosomes would carry the mutant initiator tRNA and these ribosomes would select the mutant CAT mRNA over the other mRNAs.     

Mischarging mutants of Su+2 glutamine tRNA in E. coli. II. Amino acid specificities of the mutant tRNAs

Among the mischarging mutants isolated from strains with Su+2 glutamine tRNA, two double-mutants, A37A29 and A37C38, have been suggested to insert tryptophan at the UAG amber mutation site as determined by the suppression patterns of a set of tester mutants of bacteria and phages (Yamao et al., 1988). In this paper, we screened temperature sensitive mutants of E. coli in which the mischarging suppression was abolished even at the permissive temperature. Four such mutants were obtained and they were identified as the mutants of a structural gene for tryptophanyl-tRNA synthetase (trpS). Authentic trpS mutations, such as trpS5 or trpS18, also restricted the mischarging suppression. These results strongly support the previous prediction that the mutant tRNAs of Su+2, A37A29 and A37C38, are capable of interacting with tryptophanyl-tRNA synthetase and being misaminoacylated with tryptophan in vivo. However, in an assay to determine the specificity of the mutant glutamin tRNAs, we detected predominantly glutamine, but not any other amino acid, being inserted at an amber codon in vivo to any significant degree. We conclude that the mutant tRNAs still accept mostly glutamine, but can accept tryptophan in an extent for mischarging suppression. Since the amber suppressors of Su+7 tryptophan tRNA and the mischarging mutants of Su+3 tyrosine tRNA are charged with glutamine, structural similarity among the tRNAs for glutamine, tryptophan and tyrosine is discussed.     

Anaerobic respiration of Shewanella putrefaciens requires both chromosomal and plasmid-borne genes

Abstract Pleiotropic respiratory mutants, incapable of growth on any electron acceptor other than oxygen, were isolated from two strains of Shewanella putrefaciens (MR-1 and sp200). All anaerobic respiratory functions were restored by complementation of the mutants with specific cloned DNA fragments. Southern hybridization experiments revealed that the fragment that complements the MR-1 mutant was localized on the megaplasmids of both strains, while the fragment that complements the sp200 mutant was chromosomal. Neither of these fragments hybridized with the anaerobic regulatory genes of S. putrefaciens ( etrA ) or E. coli ( fnr ).     

Suppression of amber codons in vivo as evidence that mutants derived from Escherichia coli initiator tRNA can act at the step of elongation in protein synthesis

The absence of a Watson-Crick base pair at the end of the amino acid acceptor stem is one of the features which distinguishes prokaryotic initiator tRNAs as a class from all other tRNAs. We show that this structural feature prevents Escherichia coli initiator tRNA from acting as an elongator in protein synthesis in vivo. We generated a mutant of E. coli initiator tRNA in which the anticodon sequence is changed from CAU to CUA (the T35A36 mutant). This mutant tRNA has the potential to read the amber termination codon UAG. We then coupled this mutation to others which change the C1.A72 mismatch at the end of the acceptor stem to either a U1:A72 base pair (T1 mutant) or a C1:G72 base pair (G72 mutant). Transformation of E. coli CA274 (HfrC Su- lacZ125am trpEam) with multicopy plasmids carrying the mutant initiator tRNA genes show that mutant tRNAs carrying changes in both the anticodon sequence and the acceptor stem suppress amber codons in vivo, whereas mutant tRNA with changes in the anticodon sequence alone does not. Mutant tRNAs with the above anticodon sequence change are aminoacylated with glutamine in vitro. Measurement of kinetic parameters for aminoacylation by E. coli glutaminyl-tRNA synthetase show that both the nature of the base pair at the end of the acceptor stem and the presence or absence of a base pair at this position can affect aminoacylation kinetics. We discuss the implications of this result on recognition of tRNAs by E. coli glutaminyl-tRNA synthetase.     

Novel E. coli mutants deficient in biosynthesis of 5-methylaminomethyl-2-thiouridine.

Novel E. coli mutants deficient in biosynthesis of 5- methylaminomethyl -2-thiouridine were isolated based on a phenotype of reduced readthrough at UAG codons. They define 2 new loci trmE and trmF , near 83' on the E. coli map. These mutants are different from strains carrying trmC mutations, which are known to confer a methylation deficiency in biosynthesis of 5- methylaminomethyl -2-thiouridine. tRNA from mutants carrying trmE or trmF mutations was shown to carry 2-thiouridine instead of 5- methylaminomethyl -2-thiouridine. This deficiency affects the triplet binding properties of the mutant tRNA. Our results suggest that the 5- methylaminomethyl group stabilizes the basepairing of this modified nucleotide with G, most likely through direct interaction with the ribosomal binding site(s).     

Dihydrolipoamide dehydrogenase mutation alters the NADH sensitivity of pyruvate dehydrogenase complex of Escherichia coli K-12

Under anaerobic growth conditions, an active pyruvate dehydrogenase (PDH) is expected to create a redox imbalance in wild-type Escherichia coli due to increased production of NADH (>2 NADH molecules/glucose molecule) that could lead to growth inhibition. However, the additional NADH produced by PDH can be used for conversion of acetyl coenzyme A into reduced fermentation products, like alcohols, during metabolic engineering of the bacterium. E. coli mutants that produced ethanol as the main fermentation product were recently isolated as derivatives of an ldhA pflB double mutant. In all six mutants tested, the mutation was in the lpd gene encoding dihydrolipoamide dehydrogenase (LPD), a component of PDH. Three of the LPD mutants carried an H322Y mutation (lpd102), while the other mutants carried an E354K mutation (lpd101). Genetic and physiological analysis revealed that the mutation in either allele supported anaerobic growth and homoethanol fermentation in an ldhA pflB double mutant. Enzyme kinetic studies revealed that the LPD(E354K) enzyme was significantly less sensitive to NADH inhibition than the native LPD. This reduced NADH sensitivity of the mutated LPD was translated into lower sensitivity of the appropriate PDH complex to NADH inhibition. The mutated forms of the PDH had a 10-fold-higher K(i) for NADH than the native PDH. The lower sensitivity of PDH to NADH inhibition apparently increased PDH activity in anaerobic E. coli cultures and created the new ethanologenic fermentation pathway in this bacterium. Analogous mutations in the LPD of other bacteria may also significantly influence the growth and physiology of the organisms in a similar fashion.