Expression of the ORF-2 protein of the human respiratory syncytial virus M2 gene is initiated by a ribosomal termination-dependent reinitiation mechanism

Translation of the open reading frame 2 (ORF-2) of the human respiratory syncytial virus M2 gene initiates at one of the three initiation codons located upstream of the termination codon for the first ORF. Replacement of ORF-2 with the major ORF of the chloramphenicol acetyltransferase reporter gene followed by systematic mutagenesis of the putative initiation codons demonstrated the usage of these codons as the translational initiators for ORF-2 expression both in vitro and in vivo. While the efficiency of translation was maintained when only the first and second AUG codons were preserved in vivo, there was no apparent preference in vitro for any of the three codons when only one was present. Mutagenesis studies showed that the location of the termination codon of ORF-1 protein plays a crucial role in directing translation of ORF-2 from the upstream initiation codons in vivo. This indicates that the second ORF is accessed by the ribosomes that are departing from the first ORF and that these ribosomes reinitiate on AUG codons 5' to the point of translation termination.     

Identification of strain harboring both aac(6')-Ib and aac(6')-Ib-cr variant simultaneously in Escherichia coli and Klebsiella pneumoniae

The aac(6')-Ib gene is the most prevalent gene that encodes aminoglycoside-modifying enzymes and confers resistance to tobramycin, kanamycin, and amikacin. The aac(6')-Ib-cr variant gene can induce resistance against aminoglycoside and fluoroquinolone simultaneously. Two main methods, sequence analysis and the restriction enzyme method, can detect the aac(6')-Ib-cr variant in clinical strains. We collected the 85 strains that were believed to be aac(6')-Ib positive from clinical isolates. Among them, 38 strains were the wild-type; the remaining 47 strains were the aac(6')-Ib-cr variant. Of these 47 strains, 19 simultaneously harbored aac(6')-Ib and aac(6')-Ib-cr. Our study aims to report the characteristics of the 19 strains that simultaneously harbored both genes. This study is the first investigation published in Korea of strains that included both aac(6')-Ib and aac(6')-Ib-cr variant.     

Development of an immunochromatographic assay for rapid detection of AAC(6')-Ib-producing Pseudomonas aeruginosa

To detect aminoglycoside 6'-N-acetyltransferase-Ib [AAC(6')-Ib]-producing, Pseudomonas aeruginosa isolates which are a frequent cause of nosocomial infections in Japan, an immunochromatographic assay was developed using two kinds of monoclonal antibodies (mAbs) recognizing AAC(6')-Ib. The results of the assessment were fully consistent with those of aac(6')-Ib PCR analyses.     

Biosynthesis of human fibroblast growth factor-5.

We have analyzed the biosynthesis of human fibroblast growth factor-5 (FGF-5) at the translational and posttranslational levels. FGF-5 RNA synthesized in vitro can be translated in rabbit reticulocyte lysates to yield a 29,500-Da protein, which is consistent with the molecular weight predicted from the coding sequence. The efficiency of FGF-5 translation is dramatically enhanced if an upstream open reading frame (ORF-1) in the RNA is deleted or if both AUG codons in ORF-1 are destroyed by point mutations, while partial enhancement is achieved by individual mutation of either ORF-1 AUG codon. These data suggest that FGF-5 synthesis requires the scanning of ribosomes past the two ORF-1 AUG codons. The introduction of these ORF-1 mutations into a eukaryotic FGF-5 expression vector increases its capacity to transform mouse NIH 3T3 cells up to 50-fold upon transfection. FGF-5 is secreted from transfected 3T3 cells and from human tumor cells as glycoproteins containing heterogeneous amounts of sialic acid. Glycosidase treatments suggest that the growth factor bears both N-linked and O-linked sugars.     

Genetic analysis of bacterial acetyltransferases: identification of amino acids determining the specificities of the aminoglycoside 6'-N-acetyltransferase Ib and IIa proteins.

The aminoglycoside 6'-N-acetyltransferase [AAC(6')-I] and AAC(6')-II enzymes represent a class of bacterial proteins capable of acetylating tobramycin, netilmicin, and 2'-N-ethylnetilmicin. However, an important difference exists in their abilities to modify amikacin and gentamicin. The AAC(6')-I enzymes are capable of modifying amikacin. In contrast, the AAC(6')-II enzymes are capable of modifying gentamicin. Nucleotide sequence comparison of the aac(6')-Ib gene and the aac(6')-IIa gene showed 74% sequence identity (K. J. Shaw, C. A. Cramer, M. Rizzo, R. Mierzwa, K. Gewain, G. H. Miller, and R. S. Hare, Antimicrob. Agents Chemother. 33:2052-2062, 1989). Comparison of the deduced protein sequences showed 76% identity and 82% amino acid similarity. A genetic analysis of these two proteins was initiated to determine which amino acids were responsible for the differences in specificity. Results of domain exchanges, which created hybrid AAC(6') proteins, indicated that amino acids in the carboxy half of the proteins were largely responsible for determining specificity. Mutations shifting the specificity of the AAC(6')-Ib protein to that of the AAC(6')-IIa protein (i.e., gentamicin resistance and amikacin sensitivity) have been isolated. DNA sequence analysis of four independent isolates revealed base changes causing the same amino acid substitution, a leucine to serine, at position 119. Interestingly, this serine occurs naturally at the same position in the AAC(6')-IIa protein. Oligonucleotide-directed mutagenesis was used to construct the corresponding amino acid change, a serine to leucine, in the AAC(6')-IIa protein. This change resulted in the conversion of the AAC(6')-IIa substrate specificity to that of AAC(6')-Ib. Analysis of additional amino acid substitutions within this region of AAC(6')-Ib support the model that we have identified an aminoglycoside binding domain of these proteins.     

Kinetic checkpoint at a late step in translation initiation

The translation initiation efficiency of a given mRNA is determined by its translation initiation region (TIR). mRNAs are selected into 30S initiation complexes according to the strengths of the secondary structure of the TIR, the pairing of the Shine-Dalgarno sequence with 16S rRNA, and the interaction between initiator tRNA and the start codon. Here, we show that the conversion of the 30S initiation complex into the translating 70S ribosome constitutes another important mRNA control checkpoint. Kinetic analysis reveals that 50S subunit joining and dissociation of IF3 are strongly influenced by the nature of the codon used for initiation and the structural elements of the TIR. Coupling between the TIR and the rate of 70S initiation complex formation involves IF3- and IF1-induced rearrangements of the 30S subunit, providing a mechanism by which the ribosome senses the TIR and determines the efficiency of translational initiation of a particular mRNA.     

An unstructured mRNA region and a 5' hairpin represent important elements of the E. coli translation initiation signal determined by using the bacteriophage T7 gene 1 translation start site.

Gene 1 of bacteriophage T7 early region--the RNA polymerase gene--is very actively translated during the infectious cycle of this phage. A 29 base pair fragment of its ribosome binding site containing the initiation triplet, the Shine-Dalgarno sequence (S-D), 10 nucleotides (nt) upstream and 6 nt downstream of these central elements was cloned into a vector to control the expression of the mouse dihydrofolate reductase gene (dhfr). Although all essential parts of this translation initiation region (TIR) should be present, this fragment showed only very low activity. Computer analysis revealed a potentially inhibitory hairpin binding the S-D sequence into its stem base paired to vector-derived upstream sequences. Mutational alterations demonstrated that this hairpin was not responsible for the low activity. However, addition of 21 nt of the T7 gene 1 upstream sequence to the 29 base pair fragment were capable of increasing the translational efficiency by one order of magnitude. Computer analysis of this sequence, including nucleotide shuffling, revealed that it contains a highly unstructured region lacking mRNA secondary structures but with a hairpin at its 5' end, here formed solely by T7 sequences. There was not much difference in activity whether the mRNA included or lacked vector-derived sequences upstream of the hairpin. Such highly unstructured mRNA regions were found in all very efficiently expressed T7 genes without any obvious sequence homologies. The delta G values of these regions were higher, i.e. potential secondary structural elements were fewer, than in TIR of genes from E. coli. This is likely due to the fact that T7 as a lytic phage is relying for successful infection on much stronger signals which a cell cannot afford because of the indispensable balanced equilibria of its interdependent biochemical processes. When the 5' ends of efficient T7 gene mRNA are formed by the action of RNase III they generally start with an unstructured region. Efficiently expressed T7 genes within a polycistronic mRNA, however, always contain a hairpin preceding the structure free sequence. We suggest that the formation of this 5' hairpin is releasing enough energy to keep the unstructured regions free of secondary RNA structures for sufficient time to give ribosomes and factors a good chance for binding to the TIR. In addition, sequences further downstream of the start codon give rise to an additional increase in efficiency of the TIR by almost two orders of magnitude.     

Aminoglycoside 2'-N-acetyltransferase genes are universally present in mycobacteria: characterization of the aac(2 ')-Ic gene from Mycobacterium tuberculosis and the aac(2 ')-Id gene from Mycobacterium smegmatis

The genus Mycobacterium comprises clinically important pathogens such as M. tuberculosis , which has re-emerged as a major cause of morbidity and mortality world-wide especially with the emergence of multidrug-resistant strains. The use of fast-growing species such as Mycobacterium smegmatis has allowed important advances to be made in the field of mycobacterial genetics and in the study of the mechanisms of resistance in mycobacteria. The isolation of an aminoglycoside-resistance gene from Mycobacterium fortuitum has recently been described. The aac(2 ' )-Ib gene is chromosomally encoded and is present in all isolates of M. fortuitum . The presence of this gene in other mycobacterial species is studied here and genes homologous to that of M. fortuitum have been found in all mycobacterial species studied. In this report, the cloning of the aac(2 ' )-Ic gene from M. tuberculosis H37Rv and the aac(2 ' )-Id gene from M. smegmatis mc²155 is described. Southern blot hybridizations have shown that both genes are present in all strains of this species studied to date. In addition, the putative aac(2 ' )-Ie gene has been located in a recent release of the Mycobacterium leprae genome. The expression of the aac(2 ' )-Ic and aac(2 ' )-Id genes has been studied in M. smegmatis and only aac(2 ' )-Id is correlated with aminoglycoside resistance. In order to elucidate the role of the aminoglycoside 2'- N -acetyltransferase genes in mycobacteria and to determine whether they are silent resistance genes or whether they have a secondary role in mycobacterial metabolism, the aac(2 ' )-Id gene from M. smegmatis has been disrupted in the chromosome of M. smegmatis mc²155. The disruptant shows an increase in aminoglycoside susceptibility along with a slight increase in the susceptibility to lysozyme.     

The role of bases upstream of the Shine-Dalgarno region and in the coding sequence in the control of gene expression in Escherichia coli: translation and stability of mRNAs in vivo

A range of translational initiation regions (TIR) was created by combining synthetic DNA fragments derived from the atpB-atpE intercistronic sequence of Escherichia coli with the cDNA sequence encoding mature human interleukin 2 (IL-2), the E. coli fnr gene, or an fnr::lacZ gene fusion. Both the overall rates of gene expression and the relative concentrations and stabilities of the corresponding mRNA species were estimated in strains bearing the constructs on plasmids. These measurements served as the basis for analyses of the relationship between the structure of the TIR and the true rates of translation that it promotes. The constructs involving the IL-2 cDNA were predicted to allow much less stable secondary structure within the TIR than those involving the N-terminal region of the fnr gene. Thus by combining one set of upstream sequences with two different types of N-terminal coding sequence, it was possible to distinguish between the respective influences of primary and secondary structure upon initiation. The data indicate that in the presence of a given Shine-Dalgarno (SD)/start codon combination, the decisive factor for translational initiation efficiency is the stability of base pairing involving, or in the vicinity of, this region. The sequences contributing to this secondary structure can be many bases upstream of the SD region and/or downstream of the start codon. There was no indication that the specific base sequence upstream of the SD region could, other than to the extent that it contributed to the local secondary structure, significantly influence the efficiency of translational initiation.     

Sequence and analysis of the DNA encoding protective antigen of Bacillus anthracis

The nucleotide sequence of the protective antigen (PA) gene from Bacillus anthracis and the 5' and 3' flanking sequences were determined. PA is one of three proteins comprising anthrax toxin; and its nucleotide sequence is the first to be reported from B. anthracis. The open reading frame (ORF) is 2319 bp long, of which 2205 bp encode the 735 amino acids of the secreted protein. This region is preceded by 29 codons, which appear to encode a signal peptide having characteristics in common with those of other secreted proteins. A consensus TATAAT sequence was located at the putative -10 promoter site. A Shine-Dalgarno site similar to that found in genes of other Bacillus sp. was located 7 bp upstream from the ATG start codon. The codon usage for the PA gene reflected its high A + T (69%) base composition and differed from those of genes for bacterial proteins from most other sequences examined. The TAA translation stop codon was followed by an inverted repeat forming a potential termination signal. In addition, a 192-codon ORF of unknown significance, theoretically encoding a 21.6-kDa protein, preceded the 5' end of the PA gene.     

Non-canonical translation mechanisms in plants: efficient in vitro and in planta initiation at AUU codons of the tobacco mosaic virus enhancer sequence.

The 5' untranslated leader (Omega sequence) of tobacco mosaic virus (TMV) genomic RNA was utilized as a translational enhancer sequence in expression of the 17 kDa putative movement protein (pr17) of potato leaf roll luteovirus (PLRV). In vitro translation of RNAs transcribed from appropriate chimeric constructs, as well as their expression in transgenic potato plants, resulted in the expected wild-type pr17 protein, as well as in larger translational products recognized by pr17-specific antisera. Mutational analyses revealed that the extra proteins were translated by non-canonical initiation at AUU codons present in the wild-type Omega sequence. In the plant system translation initiated predominantly at the AUU codon at positions 63-65 of the Omega sequence. Additional AUU codons in a different reading frame of the Omega sequence also showed the capacity for efficient translation initiation in vitro. These results extend the previously noted activity of the TMV 5' leader sequence in ribosome binding and translation enhancement in that the TMV translation enhancer can mediate non-canonical translation initiation in vitro and in vivo.     

Acetylation of fluoroquinolone antimicrobial agents by an Escherichia coli strain isolated from a municipal wastewater treatment plant

Aims:  To isolate environmental bacteria capable of transforming fluoroquinolones to inactive molecules.
Methods and Results:  Bacteria were isolated from the aerobic liquor of a wastewater treatment plant on a medium containing norfloxacin (100 mg l⁻¹). Twenty-two isolates were highly resistant (minimal inhibitory concentration: 6·25−200 μg ml⁻¹) to five fluoroquinolones and six of them were positive by PCR amplification for the aminoglycoside resistance gene aac(6')-Ib. Of these, only Escherichia coli strain LR09 had the ciprofloxacin-acetylating variant gene aac(6')-Ib-cr ; HPLC and mass spectrometry showed that this strain transformed both ciprofloxacin and norfloxacin by N -acetylation. This bacterium also had mutations in the quinolone-resistance determining regions of the gyrA and parC genes.
Conclusions:  An E. coli isolate from wastewater, which possessed at least two distinct fluoroquinolone resistance mechanisms, inactivated ciprofloxacin and norfloxacin by N -acetylation.
Significance and Impact of the Study:  This is the first report of N -acetylation of fluoroquinolones by an aac(6')-Ib-cr -containing bacterium from an environmental source.     

Translation initiation region sequence preferences in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

The mRNA translation initiation region (TIR) comprises the initiator codon, Shine-Dalgarno (SD) sequence and translational enhancers. Probably the most abundant class of enhancers contains A/U-rich sequences. We have tested the influence of SD sequence length and the presence of enhancers on the efficiency of translation initiation.