The transfer RNA of certain Enterobacteriacae contain 2-methylthiozeatin riboside (ms2io6A) an isopentenyl adenosine derivative

Isopentenyl adenosine derivatives are always located adjacent to the 3' end of the anticodon in transfer RNA and have been implicated in certain biological functions. In the enteric bacterium, E. coli, the derivative is ms2i6A whereas in some plant associated bacteria the derivative is the hydroxylated form, ms2io6A. Anti-i6A immunoadsorbent chromatography has been employed to detect isopentenyl adenosine compounds. In the present study we show that the transfer RNA of three species of enteric bacteria, S. typhimurium, K. pneumoniae, and S. marcescens contains both ms2io6A and ms2i6A. Under the growth conditions utilized the ms2io6A is predominant. The presence of ms2io6A in Enterobacteriacae is particularly noteworthy since in previous work it has been found only in plant-associated species of bacteria.     

The effect of point mutations affecting Escherichia coli tryptophan tRNA on anticodon-anticodon interactions and on UGA suppression

tRNA species in Escherichia coli that translate codons starting with U contain 2-methyl-thio-N6-isopentenyl-adenosine in position 37, 3' adjacent to the anticodon. The role of this hypermodification in protein synthesis and trp operon attenuation has been investigated. Temperature-jump relaxation methods have been applied to study the interaction between E. coli tRNAPro, with anticodon VGG (V is uridine-5-oxyacetic acid) complementary to that of tRNATrp, and three species of E. coli tRNATrp: wild type tRNATrp (with ms2i6A37 and G24), UGA suppressor tRNATrp (with ms2i6A37 and A24 in the dihydrouridine stem but the same anticodon CCA), and the same suppressor molecule but ms2i6A-deficient as a result of the mutation miaA. Complex formation between tRNAPro and ms2i6A-containing tRNATrp shows thermodynamic parameters close to those found for several other pairs of tRNA with complementary anticodons. However, ms2i6A-deficient tRNATrp makes less stable complexes with tRNAPro, which dissociate eightfold faster. No effect on the complementary anticodon interaction of the mutation in the dihydrouridine stem can be detected. When the tRNA analogous to the opal codon, E. coli tRNASerIV (anticodon VGA) replaces tRNAPro in similar experiments, very weak complexes are observed with both normally hypermodified species of tRNATrp, the wild type and UGA suppressor; these show a lifetime about 50-fold shorter than with tRNAPro, but are again similar. No complex formation is detectable with the ms2i6A-deficient species. This may explain why the hypermodification is necessary for the efficient suppression of the UGA terminator of Q beta coat protein in vitro. The data on complexes with tRNAPro suggest that deficiency in ms2i6A may also reduce the efficiency of UGG reading. Thus, miaA may affect trp operon attenuation by slowing translation of the tandem UGG codons in the leader sequence. Temperature-jump differential spectra suggest that ms2i6 stabilizes the anticodon interaction by improved stacking of base 37.     

cis 2-Methylthio-ribosylzeatin (ms2io6A) is present in the transfer RNA of Salmonella typhimurium, but not Escherichia coli

We have identified the cis isomer of N6-(4-hydroxy-isopentenyl)-2-methylthioadenosine (ms2io6A) as a component of the tRNA of Salmonella typhimurium. This is the first report of this compound in the tRNA of any member of the enterobacteriaceae: the nucleoside was previously thought to be found exclusively in plants or plant associated bacteria. Interestingly, all E. coli strains examined were found to lack ms2io6A. Evidence is presented which suggests S. typhimurium tRNA also contains low levels of 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U) in addition to 5-methylaminomethyl-2-thiouridine (mnm5s2U).     

Influence of modification next to the anticodon in tRNA on codon context sensitivity of translational suppression and accuracy.

Effects on translation in vivo by modification deficiencies for 2-methylthio-N6-isopentenyladenosine (ms2i6A) (Escherichia coli) or 2-methylthio-N6-(4-hydroxyisopentenyl)adenosine (ms2io6A) (Salmonella typhimurium) in tRNA were studied in mutant strains. These hypermodified nucleosides are present on the 3' side of the anticodon (position 37) in tRNA reading codons starting with uridine. In E. coli, translational error caused by tRNA was strongly reduced in the case of third-position misreading of a tryptophan codon (UGG) in a particular codon context but was not affected in the case of first-position misreading of an arginine codon (CGU) in another codon context. Misreading of UGA nonsense codons at two different positions was codon context dependent. The efficiencies of some tRNA nonsense suppressors were decreased in a tRNA-dependent manner. Suppressor tRNA which lacks ms2i6A-ms2io6A becomes more sensitive to codon context. Our results therefore indicate that, besides improving translational efficiency, ms2i6A37 and ms2io6A37 modifications in tRNA are also involved in decreasing the intrinsic codon reading context sensitivity of tRNA. Possible consequences for regulation of gene expression are discussed.     

The methylthio group (ms2) of N6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms2io6A) present next to the anticodon contributes to the decoding efficiency of the tRNA.

A Salmonella typhimurium LT2 mutant which harbors a mutation (miaB2508::Tn10dCm) that results in a reduction in the activities of the amber suppressors supF30 (tRNA(CUATyr)), supD10 (tRNA(CUASer)), and supJ60 (tRNA(CUALeu)) was isolated. The mutant was deficient in the methylthio group (ms2) of N6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms2io6A), a modified nucleoside that is normally present next to the anticodon (position 37) in tRNAs that read codons that start with uridine. Consequently, the mutant had i6A37 instead of ms2io6A37 in its tRNA. Only small amounts of io6A37 was found. We suggest that the synthesis of ms2io6A occurs in the following order: A-37-->i6A37-->ms2i6A37-->ms2io6A37. The mutation miaB2508::Tn10dCm was 60% linked to the nag gene (min 15) and 40% linked to the fur gene and is located counterclockwise from both of these genes. The growth rates of the mutant in four growth media did not significantly deviate from those of a wild-type strain. The polypeptide chain elongation rate was also unaffected in the mutant. However, the miaB2508::Tn10dCm mutation rendered the cell more resistant or sensitive, compared with a wild-type cell, to several amino acid analogs, suggesting that this mutation influences the regulation of several amino acid biosynthetic operons. The efficiencies of the aforementioned amber suppressors were decreased to as low as 16%, depending on the suppressor and the codon context monitored, demonstrating that the ms2 group of ms2io6A contributes to the decoding efficiency of tRNA. However, the major impact of the ms2io6 modification in the decoding process comes from the io6 group alone or from the combination of the ms2 and io6 groups, not from the ms2 group alone.     

ms2i6A deficiency enhances proofreading in translation.

The hypermodified base 2-methylthio-N6-isopentenyladenosine (ms2i6A) at position 37 occurs frequently in tRNAs that read codons starting with uridine. Here we have studied how ms2i6A affects the accuracy of poly(U) translation in vitro. Deficiency leads to a higher rejection rate of tRNA4(Leu) by more aggressive proofreading on the wild-type ribosome, but with the initial selection step unchanged. Our data indicate that ms2i6A has no effect on codon-anticodon interactions on wild-type ribosomes as long as aminoacyl-tRNA is in ternary complex with EF-Tu and GTP. ms2i6A deficiency in the cognate poly(U) reader tRNA(Phe) leads to increased misreading when the near-cognate competitor tRNA4(Leu) is wild-type. ms2i6A deficiency in tRNA4(Leu) gives a decreased error level in competition with wild-type tRNA(Phe).     

Synthesis of a Klebsiella pneumoniae O-antigen heteropolysaccharide (O12) requires an ABC 2 transporter

A recombinant clone encoding enzymes for Klebsiella pneumoniae O12-antigen lipopolysaccharide (LPS) was found when we screened for serum resistance of a cosmid-based genomic library of K. pneumoniae KT776 (O12:K80) introduced into Escherichia coli DH5alpha. A total of eight open reading frames (ORFs) (wb(O12) gene cluster) were necessary to produce K. pneumoniae O12-antigen LPS in E. coli K-12. A complete analysis of the K. pneumoniae wb(O12) cluster revealed an interesting coincidence with the wb(O4) cluster of Serratia marcescens from ORF5 to ORF8 (or WbbL to WbbA). This prompted us to generate mutants of K. pneumoniae strain KT776 (O12) and to study complementation between the two enterobacterial wb clusters using mutants of S. marcescens N28b (O4) obtained previously. Both wb gene clusters are examples of ABC 2 transporter-dependent pathways for O-antigen heteropolysaccharides. The wzm-wzt genes and the wbbA or wbbB genes were not interchangeable between the two gene clusters despite their high level of similarity. However, introduction of three cognate genes (wzm-wzt-wbbA or wzm-wzt-wbbB) into mutants unable to produce O antigen allowed production of the specific O antigen. The K. pneumoniae O12 WbbL protein performs the same function as WbbL from S. marcescens O4 in either the S. marcescens O4 or E. coli K-12 genetic background.     

Purification and Partial Characterization of the B Subunit of Serratia marcescens Tryptophan Synthetase

A trpE mutant of Serratia marcescens (E-7) was isolated, and the multimeric enzyme tryptophan synthetase (EC 4.2.1.20) was purified to homogeneity from derepressed cells. The A and B subunits were resolved, and the B subunit was partially characterized and compared with the Escherichia coli B subunit as part of a comparative evolution study of the trpB cistron of the trp operon in the Enterobacteriaceae. The S. marcescens B subunit is a dimer (beta(2)), and its molecular weight was estimated to be 89,000. The separate subunits (beta monomers) had molecular weights of approximately 43,000. The B subunit required pyridoxal phosphate for catalytic activity and had an apparent K(m) of 9 x 10(-6) M. The N terminus of the B subunit was unavailable for reaction with terminal amine reagents (blocked), whereas carboxypeptidase digestion released a C-terminal isoleucine. Using S. marcescens B antiserum in agar immunodiffusion gave an almost complete reaction of identity between the B subunits of S. marcescens and E. coli. The antiserum was used in microcomplement fixation, allowing for a comparison of the overall antigenic surface structure of the two B subunits. The index of dissimilarity for the heterologous E. coli enzyme compared with the homologous S. marcescens enzyme was 2.4, indicating extensive similarity of the two proteins at their surfaces. Comparative antiserum neutralization of B-subunit enzyme activity showed the E. coli enzyme to cross-react 85% as well as the S. marcescens enzyme. With regard to the biochemical and immunochemical parameters used in this study, the S. marcescens and E. coli B subunits were either identical or very similar. These findings support the idea that the trpB cistron of the trp operon is a relatively conserved gene in the Enterobacteriaceae.     

Isolation and Characterization of Specialized φ80 Transducing Phages Carrying Regions of the Salmonella typhimurium trp Operon

We have isolated a series of nondefective phi80 specialized transducing phage which carry segments of the Salmonella typhimurium trp operon. These phage were obtained from a lysogenic derivative of a merozygote constructed by transferring an S. typhimurium trp episome into an Escherichia coli strain which lacks the normal phi80 attachment site. The deoxyribonucleic acid (DNA) from one such phage was purified and employed in DNA-ribonucleic acid (RNA) hybridization studies. The results obtained show that, under our hybridization conditions, heterologous hybridization is less efficient than homologous hybridization. It was also observed that not all S. typhimurium trp messenger RNA can readily anneal to E. coli trp operon DNA. Heterologous hybrids consisting of S. typhimurium trp messenger RNA and E. coli trp operon DNA were estimated to have a dissociation constant 10-fold larger than that of homologous hybrids.     

The occurrence of duplicate lysyl-tRNA synthetase gene homologs in Escherichia coli and other procaryotes.

The lysyl-tRNA synthetase (LysRS) system of Escherichia coli K-12 consists of two genes, lysS, which is constitutive, and lysU, which is inducible. It is of importance to know how extensively the two-gene LysRS system is distributed in procaryotes, in particular, among members of the family Enterobacteriaceae. To this end, the enterics E. coli K-12 and B; E. coli reference collection (ECOR) isolates EC2, EC49, EC65, and EC68; Shigella flexneri; Salmonella typhimurium; Klebsiella pneumoniae; Enterobacter aerogenes; Serratia marcescens; and Proteus vulgaris and the nonenterics Pseudomonas aeruginosa and Bacillus megaterium were grown in AC broth to a pH of 5.5 or less or cultured in SABO medium at pH 5.0. These growth conditions are known to induce LysRS activity (LysU synthesis) in E. coli K-12. Significant induction of LysRS activity (twofold or better) was observed in the E. coli strains, the ECOR isolates, S. flexneri, K. pneumoniae, and E. aerogenes. To demonstrate an association between LysRS induction and two distinct LysRS genes, Southern blotting was performed with a probe representing an 871-bp fragment amplified from an internal portion of the coding region of the lysU gene. In initial experiments, chromosomal DNA from E. coli K-12 strain MC4100 (lysS+ lysU+) was double digested with either BamHI and HindIII or BamHI and SalI, producing hybridizable fragments of 12.4 and 4.2 kb and 6.6 and 5.2 kb, respectively. Subjecting the chromosomal DNA of E. coli K-12 strain GNB10181 (lysS+ delta lysU) to the same regimen established that the larger fragment from each digestion contained the lysU gene. The results of Southern blot analysis of the other bacterial strains revealed that two hybridizable fragments were obtained from all of the E. coli and ECOR collection strains examined and S. flexneri, K. pneumoniae, and E. aerogenes. Only one lysU homolog was found with S. typhimurium and S. marcescens, and none was obtained with P. vulgaris. A single hybridizable band was found with both P. aeruginose and B, megaterium. These results show that the dual-gene LysRS system is not confined to E. coli K-12 and indicate that it may have first appeared in the genus Enterobacter.     

Investigation of immunoadsorbent efficiency and capacity for the isolation of tRNAs containing N6-(delta 2-isopentenyl)adenosine derivatives

Antibodies directed to modified nucleosides recognize the nucleoside (antigen) when it is present in an intact tRNA molecule. The general application of anti-nucleoside immunoadsorbent chromatography, however, has been greatly impeded by the apparent inefficiency and low capacity of conventional immunoadsorbents for transfer RNA. Antibodies specific for isopentenyladenosine (i6A) were employed to investigate the efficacy of various immunoadsorbents with respect to immobilization of antibody protein and with respect to their ability to bind i6A-containing tRNAs. Biologically active anti-i6A was recovered in high yield (80-88%) by affinity chromatography on i6A-adipate-Sepharose 4B or i6A-butane diglycidyl ether-Sepharose 4B using either 15% pyridine in phosphate-buffered saline or 0.2 M acetic acid as eluents. The binding capacity of various anti-i6A antibody immunoadsorbents was evaluated. While both anti-i6A antibody-protein A-agarose-iminothiolane (ITL) and anti-i6A antibody-protein A-agarose-dimethyl suberimidate showed a high capacity for i6A-tRNA, the latter column is much less efficient with respect to antibody immobilization. Under optimal conditions, the ITL immunoadsorbent binds 5-6 nmol of i6A/mg of antibody protein. With respect to bulk tRNA, 1 mg of antibody protein (ITL immunoadsorbent) binds all of the i6A-tRNA in a 1-mg sample.     

Geosmin and 2-methylisoborneol from cyanobacteria in three water supply systems

The changes in populations of Staphylococcus aureus, Bacillus subtilis, Salmonella typhimurium, Klebsiella pneumoniae, Agrobacterium tumefaciens, Rhizobium meliloti, and Saccharomyces cerevisiae were measured after their introduction into samples of sewage, lake water, and soil. Enumeration of small populations was possible because the strains used were resistant to antibiotics in concentrations and combinations such that few species native to these ecosystems were able to grow on agar containing the inhibitors. Fewer than 2 cells per ml of sewage or lake water and 25 cells per g of soil could be detected. A. tumefaciens and R. meliloti persisted in significant numbers with little decline, but S. aureus, K. pneumoniae, S. typhimurium, S. cerevisiae, and vegetative cells of B. subtilis failed to survive in samples of sewage and lake water. In sterile sewage, however, K. pneumoniae, B. subtilis, S. typhimurium, A. tumefaciens, and R. meliloti grew; S. cerevisiae populations were maintained at the levels used for inoculation; and S. aureus died rapidly. In sterile lake water, the population of S. aureus and K. pneumoniae and the number of vegetative cells of B. subtilis declined rapidly, R. meliloti grew, and the other species maintained significant numbers with little or a slow decline. The populations of S. aureus, K. pneumoniae, A. tumefaciens, B. subtilis, and S. typhimurium declined in soil, but the first four species grew in sterile soil. It is suggested that some species persist in environments in which they are not indigenous because they tolerate abiotic stresses, do not lose viability readily when starved, and coexist with antagonists. The species that fails to survive need only be affected by one of these factors.     

Immunospecific binding of tRNA precursors containing N6-(delta 2-isopentenyl) adenosine

Antibodies specific for N6-(delta 2-isopentenyl) adenosine (i6A) were immobilized on Sepharose and this adsorbent (Sepharose-anti-i6A) was used to selectively isolate bacteriophage T4 tRNA precursors containing i6A/ms2i6A from an unfractionated population of 32P-labeled T4 RNAs. The results showed that antibodies to i6A selectively bound only those tRNA precursors containing i6A/ms2i6A. Binding of tRNA precursors by antibody and specificity of the binding was assessed by membrane binding using 32P-labeled tRNA precursor. Binding was highly specific for i6A/ms2i6A residues in the tRNA precursors. This binding can be used to separate modified from unmodified precursor RNAs and to study the biosynthetic pathways of tRNA precursors.     

Immunological investigation of the distribution of cytochromes related to the two terminal oxidases of Escherichia coli in other gram-negative bacteria.

Monospecific antibodies were raised against the two terminal oxidase complexes of the aerobic respiratory chain of Escherichia coli. These are the cytochrome d and cytochrome o complexes. The antibodies were used to check for the occurrence of cross-reactive antigens in membrane preparations from a variety of gram-negative bacteria by rocket immunoelectrophoresis and immunoblotting techniques. With these criteria, proteins closely related to the cytochrome d complex of E. coli appeared to be widely distributed. Among the strains containing cytochrome d-related material were Serratia marcescens, Photobacterium phosphoreum, Salmonella typhimurium, Klebsiella pneumoniae, and Azotobacter vinelandii. The data suggest that the d-type terminal oxidase in many of these strains is associated in a complex with b-type and a1-type cytochromes, as has been found to be the case in E. coli. K. pneumoniae and S. typhimurium were also shown to have material cross-reactive to the E. coli cytochrome o complex.     

Alternative prey: a mechanism for elimination of bacterial species by protozoa

Antibiotic-resistant strains of Salmonella typhimurium and Klebsiella pneumoniae died readily after their addition to raw sewage, but they grew in sterilized sewage. The decline was not a result of abiotic stresses, and because the bacteria were able to survive in large numbers for at least 15 days in solutions containing no organic nutrients, it was not a result of competition. Toxin production, bacteriophages, and Bdellovibrio sp. did not cause the disappearance of the two bacterial species. A decline was also evident if the sewage was first passed through a 3-micron (pore size) filter or treated with cycloheximide or cycloheximide plus nystatin, but protozoa developed under these conditions. Little or no decline occurred if the sewage was filtered and treated with the eucaryotic inhibitors before the addition of S. typhimurium or K. pneumoniae, and protozoa were not detected. S. typhimurium increased in abundance if cycloheximide, streptomycin, and erythromycin or large amounts of glucose were added to sewage. Tetrahymena thermophilus did not significantly reduce the population of S. typhimurium in buffer when the density of the bacterium was about 10(4)/ml. However, when more than 10(8) Enterobacter agglomerans cells per ml were added to the buffer, T. thermophilus reduced the abundance of E. agglomerans and S. typhimurium to 10(6) and 10/ml, respectively. The density of S. typhimurium was further decreased by a second increment of E. agglomerans cells. The disappearance of S. typhimurium and K. pneumoniae from sewage thus is the result of predation by protozoa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transfer of episome F'lac+ and chromosomal trp+ genes from Erwinia amylovora to Salmonella typhimurium.

The F'lac+ episome of Escherichia coli origin was transferred by conjugation with frequencies of 10(-7) to 10(-5) from Erwinia amylovora to 14 out of 15 Salmonella typhimurium trp female parents. The chromosomal trp+ genes were transferred with frequencies of 10(-7) to 10(-6) only to one trpB and 2 trpD female parents, which have a point mutation in the 2nd and fourth structural genes, respectively, of the tryptophan operon. The transferred male trp+ genes became integrated at the selected sites of the S. tryphimurium chromosome. The resulting Trp+ hybrids were phenotypically stable, lacked a cryptic trp allele selected against in the female parent, had high genetic homology values in the tryptophan region, and showed biochemical reactions and pathogenicity typical of S. typhimurium.     

A modified nucleotide in tRNA as a possible regulator of aerobiosis: synthesis of cis-2-methyl-thioribosylzeatin in the tRNA of Salmonella

The state of modification of the adenosine residue (A37), found adjacent to the anticodon in tRNAs that recognize codons beginning with U, varies in Salmonella bacteria grown under different physiological conditions. In aerobically grown bacteria, these tRNAs contain ms2io6A and in bacteria grown anaerobically they contain its precursor, ms2i6A. The hydroxylation of the isopentenyl (i6-) side chain of ms2i6A does not occur in the absence of oxygen. When the bacteria are grown under iron or cysteine limitation the tRNAs contain predominantly i6A, rather than ms2i6A, ms2io6A, or io6A. The bacteria do not methylthiolate (ms2-) the i6A under these conditions. A Salmonella miaA mutant lacking the isopentenylation enzyme contains an A37 rather than any of the modified forms. Some of the biosynthetic pathways of the amino acids corresponding to ms2i6A containing tRNAs (phe, tyr, trp, ser, leu, cys) are known to have altered regulation depending on the state of modification of nucleoside A37. This regulation appears to be effected through attenuation. We hypothesize that these varying states of modification are related to electron-acceptor pathways in anaerobic or aerobic growth. The role of ms2io6-adenine (the cytokinin hormone in plants) and i6-adenine (an activator of the cell cycle in animal cells) is discussed as related to the role of modifying enzymes in regulation.