Cointegrational transduction and mobilization of gentamicin resistance plasmid pWP14a is mediated by IS140

The structures of two R-plasmids pWP14a and pWP12a (Tra-, Ap, Gm; 21 kb) and of several cointegrates they form with bacteriophages P1Cm and P1-15 were analyzed. In each case, replicon fusion was mediated by the element IS140 (about 0.8 kb), one copy of which resides on both plasmids adjacent to the gentamicin resistance determinant (AAC(3)-III). pWP14a cointegrated preferentially into or near the invertible C-loop structure of the P1 genome. Cointegrational mobilization of pWP14a was observed also with several conjugative R-factors. The process of replicon fusion is independent of the host's rec+ functions. Sequences homologous to IS140 are constituents of many R-factors, including RA1, R40a, R124, R144, Rts1, N3, and pJR255. IS140 also shows homology to two other sequences, IS15 delta and Tn2680, but not to other, well studied transposable elements. The ampicillin resistance determinant of pWP14a is within a Tn3-like transposon, Tn3651.     

Development of a plant transformation selection system based on expression of genes encoding gentamicin acetyltransferases

The development of selectable markers for transformation has been a major factor in the successful genetic manipulation of plants. A new selectable marker system has been developed based on bacterial gentamicin-3-N-acetyltransferases [AAC(3)]. These enzymes inactivate aminoglycoside antibiotics by acetylation. Two examples of AAC(3) enzymes have been manipulated to be expressed in plants. Chimeric AAC(3)-III and AAC(3)-IV genes were assembled using the constitutively expressed cauliflower mosaic virus 35S promoter and the nopaline synthase 3′ nontranslated region. These chimeric genes were engineered into vectors for Agrobacterium-mediated plant transformation. Petunia hybrida and Arabidopsis thaliana tissue transformed with these vectors grew in the presence of normally lethal levels of gentamicin. The transformed nature of regenerated Arabidopsis plants was confirmed by DNA hybridization analysis and inheritance of the selectable phenotype in progeny. The chimeric AAC(3)-IV gene has also been used to select transformants in several additional plant species. These results show that the bacterial AAC(3) genes will serve as useful selectable markers in plant tissue culture.     

A molecular-genetic study of the Arabidopsis Toc75 gene family

Toc75 (translocon at the outer envelope membrane of chloroplasts, 75 kD) is the protein translocation channel at the outer envelope membrane of plastids and was first identified in pea (Pisum sativum) using biochemical approaches. The Arabidopsis (Arabidopsis thaliana) genome contains three Toc75-related sequences, termed atTOC75-I, atTOC75-III, and atTOC75-IV, which we studied using a range of molecular, genetic, and biochemical techniques. Expression of atTOC75-III is strongly regulated and at its highest level in young, rapidly expanding tissues. By contrast, atTOC75-IV is expressed uniformly throughout development and at a much lower level than atTOC75-III. The third sequence, atTOC75-I, is a pseudogene that is not expressed due to a gypsy/Ty3 transposon insertion in exon 1, and numerous nonsense, frame-shift, and splice-junction mutations. The expressed genes, atTOC75-III and atTOC75-IV, both encode integral envelope membrane proteins. Unlike atToc75-III, the smaller atToc75-IV protein is not processed upon targeting to the envelope, and its insertion does not require ATP at high concentrations. The atTOC75-III gene is essential for viability, since homozygous atToc75-III knockout mutants (termed toc75-III) could not be identified, and aborted seeds were observed at a frequency of approximately 25% in the siliques of self-pollinated toc75-III heterozygotes. Homozygous toc75-III embryos were found to abort at the two-cell stage. Homozygous atToc75-IV knockout plants (termed toc75-IV) displayed no obvious visible phenotypes. However, structural abnormalities were observed in the etioplasts of toc75-IV seedlings and atTOC75-IV overexpressing lines, and toc75-IV plants were less efficient at deetiolation than wild type. These results suggest some role for atToc75-IV during growth in the dark.     

An exotype alginate lyase in Sphingomonas sp. A1: overexpression in Escherichia coli,purification, and characterization of alginate lyase IV (A1-IV)

Sphingomonas sp. A1 (strain A1) cells contain three kinds of endotype alginate lyases [A1-I, A1-II, and A1-III], all of which are formed from a common precursor through posttranslational processing. In addition to these lyases, another type of lyase (A1-IV) that acts on oligoalginates exists in the bacterium. A1-IV was overexpressed in Escherichia coli cells through control of its gene under the T7 promoter. The expression level of the enzyme in E. coli cells was 8.6U/L-culture, which was about 270-fold higher than that in strain A1 cells. The enzyme was purified to homogeneity through three steps with an activity yield of 10.9%. The optimal pH and temperature, thermal stability, and mode of action of the purified enzyme were similar to those of the native enzyme from strain A1 cells. A1-IV exolytically degraded oligoalginates, which were produced from alginate through the reaction of A1-I, A1-II, or A1-III, into monosaccharides, indicating that the cooperative actions of these four enzymes cause the complete depolymerization of alginate in strain A1 cells.     

Aminoglycoside-inactivating enzymes in strains of Salmonella and genes encoding them

Aminoglycoside resistance patterns of 56 strains isolated from man, cattle and environment were determined. 34 out of 42 gentamicin-resistant strains were shown to produce AAC(3)-II and 7 strains produced ANT(2"). All the 48 kanamycin resistant strains produced APH(3')-I. Spot hybridization of the 42 gentamicin resistant strains with the inner fragment of the aacC2 gene revealed positive signals for all the strains. Hybridization of the 48 kanamycin-resistant strains with the aphA1 gene probe provided positive results in all the strains. The AAC(3)-IV encoding gene was not detected by DNA-DNA hybridization in the strains studied.     

The kinetic mechanism of AAC3-IV aminoglycoside acetyltransferase from Escherichia coli

The aminoglycoside 3-N-acetyltransferase AAC(3)-IV from Escherichia coli exhibits a very broad aminoglycoside specificity, causing resistance to a large number of aminoglycosides, including the atypical veterinary antibiotic, apramycin. We report here on the characterization of the substrate specificity and kinetic mechanism of the acetyl transfer reaction catalyzed by AAC(3)-IV. The steady-state kinetic parameters revealed a narrow specificity for the acyl-donor and broad range of activity for aminoglycosides. AAC(3)-IV has the broadest substrate specificity of all AAC(3)'s studied to date. Dead-end inhibition and ITC experiments revealed that AAC(3)-IV follows a sequential, random bi-bi kinetic mechanism. The analysis of the pH dependence of the kinetic parameters revealed acid- and base-assisted catalysis and the existence of three additional ionizable groups involved in substrate binding. The magnitude of the solvent kinetic isotope effects suggests that a chemical step is at least partially rate limiting in the overall reaction.     

Detection of apramycin resistant enterobacteriaceae in hospital isolates

Apramycin is a recently developed aminoglycoside restricted to veterinary therapy. Production of a 3-aminoglycoside acetyltransferase type IV (AAC(3)-IV) conferring cross-resistance to this drug and to gentamicin was detected in 1984 in France in bacteria of bovine origin. This mechanism of resistance was apparently confined to animals. We have studied 17 strains resistant to apramycin and gentamicin isolated in 5 hospitals in Belgium. Conjugative plasmids encoding an AAC(3)IV were present in 14 isolates. Comparison of the restriction fingerprints revealed 6 different plasmid patterns: 8 plasmids belonged to 2 groups sharing extensive intragroup homology and 4 were not related to the other replicons. These results indicate dissemination of plasmids within and between hospitals, but also of the gene encoding an AAC(3)IV.     

Evolutionary changes in the structural and regulatory regions of aminoglycoside-3'-phosphotransferase type I gene of E. coli

To investigate the evolutionary relationships between the aph(3') genes from different plasmids, the nucleotide sequence of the aph(3') gene from the E. coli R plasmid was determined and compared with the known aph(3') genes of Tn903 and Tn4352. Three point mutations in the structural part of the cloned aph(3') gene caused amino acid changes in the enzyme molecule at positions 19, 27 and 48 beginning from the start codon. The structural part of the gene was followed by two stop codons and a long DNA region containing no nucleotide sequences homologous to the sequences of Tn903 or Tn4352. Both the cloned aph(3') gene and Tn4352 were limited on the left by the spacer sequence and the insertion sequence IS176. Twenty one base pairs deletion abolished the -35 sequence of the promoter suggested for the aph(3') gene of Tn4352 and resulted in formation of a fusion promoter utilizing the -35 box of IS176 and the -10 box of the aph(3') gene. The distance between the -35 and -10 sequences changed from 18 to 17 bp. Changes in the cloned aph(3') gene and the flanking DNA regions resulted in formation of a new promoter and loss of the right IS176 element.     

Prevalence of IS(Aba1) in epidemiologically unrelated Acinetobacter baumannii clinical isolates

Seventy-five Acinetobacter baumannii strains belonging to different pulsetypes, plus one ceftazidime-susceptible strain, from a pulsetype in which all strains were resistant, were included in this study. The minimum inhibitory concentration of ceftazidime was determined by the microdilution method. The bla(ADC)-like gene, the IS(Aba1) element and the IS(Aba1) located in the bla(ADC)-like promoter were detected by PCR. The objective of the study was to determine the prevalence of IS(Aba1) in a collection of epidemiologically unrelated A. baumannii clinical isolates. The bla(ADC)-like gene was detected in 74 (97.3%) out of the 76 strains analysed. In these 74 strains, 51 (69%) were positive for the IS element and it was not detected in 23 (31%) strains. Among the A. baumannii strains containing the IS element, 40 (78.4%) had the IS element located in the promoter region of the bla(ADC)-like gene. In a high percentage of A. baumannii clinical isolates carrying the IS(Aba1), this is inserted into the promoter region of the bla(ADC)-like gene. In addition, two clinical isolates belonging to the same pulsetype, one with and one without the IS(Aba1), can be found in the clinical setting, suggesting the potential acquisition or loss of this genetic element in the hospital environment.     

Primary structure of an aminoglycoside 6''-N-acetyltransferase AAC(6'')-4, fused in vivo with the signal peptide of the Tn3-encoded beta-lactamase.

The gene aacA4 encoding an aminoglycoside 6'-N-acetyltransferase, AAC(6')-4, was cloned from a natural multiresistance plasmid, and its nucleotide sequence was determined. The gene was 600 base pairs (bp) long, and the AAC(6')-4 had a calculated molecular size of 22.4 kilodaltons and an isoelectric point of 5.35. The sequence of the 17 N-terminal amino acids was determined from the purified enzyme. The AAC(6')-4 gene was part of a resistance gene cluster, and its expression was under the control of the regulatory sequences of the beta-lactamase encoded by Tn3. The five N-terminal amino acids were identical to those of the signal peptide of the Tn3-encoded beta-lactamase, and the entire 5' region of aacA4, as far as it was sequenced (354 bp, including the promoter and the ribosome-binding site sequences), was identical to that of the beta-lactamase gene. This led us to presume an in vivo fusion between the beta-lactamase and the acetyltransferase genes. The latter was followed, in a polycistronic arrangement, by an aminoglycoside 3",9-adenylyltransferase gene, aadA, with an intergenic region of 68 bp. At a distance of ca. 1.3 kilobases in the 3' direction, we found remnants of a second Tn3-like element specifying an active beta-lactamase. At their 5' extremities, the two incomplete copies of Tn3, which were in tandem orientation, were interrupted within the resolvase gene. We speculate that Tn3-related sequences have played a role in the process of selection and dissemination of the AAC(6')-4 gene, which specifies resistance to amikacin and related aminoglycosides.     

Genetic organization of insertion element IS2 based on a revised nucleotide sequence

We identified a transposable element resident in the chromosome of Escherichia coli K-12 strain HB101. This is an approx. 4400-bp-long transposon flanked by two copies of insertion sequence (IS) 1 element in direct orientation. One of the IS1 elements was found to be integrated into an IS2 element between IS2 bp 139 and bp 140 with the large moiety of IS2 within the transposon. The sequence of this part of IS2 differs from the published sequence of galOP-308::IS2 at a number of positions. Restriction analysis of the published allele, however, indicated that both alleles may in fact be identical. Since six of the eight differences found alter open reading frames, the revised sequence presents a new outlook for the potential genetic organization of IS2.     

The effects of dietary lipid and phenobarbitone on the production and utilization of NADPH in the liver. A combined biochemical and quantitative cytochemical study.

The unit A-type glycopeptides were purified from porcine thyroglobulin by Pronase digestion followed by chromatography on a DEAE-Sephadex A-25 column. These glycopeptides were separated into five fractions (UA-I, -II, -IV and -V) by Dowex 50W (X2) column chromatography. Fractions UA-I, -II, -III, -IV and -V were found to have the compositions (Man)9(GlcNAc)2-Asn, (Man)8(GlcNAc)2-Asn, (Man)7(GlcNAc)2-Asn, (Man)6(GlcNAc)2-Asn and (Man)5(GlcNAc)2-Asn respectively. The structures of these five fractions were investigated by the combination of exo- and endo-glycosidase digestions, methylation analysis. Smith periodate degradation and acetolysis. The results showed that fraction UA-V had the simplest structure: see formula in text. The larger glycopeptides (fractions UA-I, -II, -III and -IV) contained additional mannose residues alpha (1 leads to 2)-linked to the terminal mannose residues in the above core structure. These unit A-type glycopeptides appear to be biosynthetic intermediates that are to be processed to form complex-type glycopeptides (unit B-type sugar chains).     

Conservation of Position and Sequence of a Novel, Widely Expressed Gene Containing the Major Human α-Globin Regulatory Element

We have determined the cDNA and genomic structure of a gene (−14 gene) that lies adjacent to the human α-globin cluster. Although it is expressed in a wide range of cell lines and tissues, a previously described erythroid-specific regulatory element that controls expression of the α-globin genes lies within intron 5 of this gene. Analysis of the −14 gene promoter shows that it is GC rich and associated with a constitutively expressed DNase 1 hypersensitive site; unlike the α-globin promoter, it does not contain a TATA or CCAAT box. These and other differences in promoter structure may explain why the erythroid regulatory element interacts specifically with the α-globin promoters and not the −14 gene promoter, which lies between the α promoters and their regulatory element. Interspecies comparisons demonstrate that the sequence and location of the −14 gene adjacent to the α cluster have been maintained since the bird/mammal divergence, 270 million years ago.