Loss of intrinsic aminoglycoside resistance in Acinetobacter haemolyticus as a result of three distinct types of alterations in the aac(6')-Ig gene, including insertion of IS17

The distribution of the aac(6')-Ig gene, encoding aminoglycoside 6'-N-acetyltransferase-Ig [AAC(6')-Ig], was studied in 96 Acinetobacter haemolyticus strains and 12 proteolytic Acinetobacter strains, including Acinetobacter genomospecies 6, 13, and 14 and 3 unnamed species assigned to this genomic group by DNA-DNA hybridization. This gene was detected by DNA-DNA hybridization in all 96 A. haemolyticus strains and by PCR in 95 strains but was not detected in strains of other species, indicating that it may be used to identify A. haemolyticus. Three A. haemolyticus strains were susceptible to tobramycin and did not produce an aminoglycoside 6'-N-acetylating activity, although they contained aac(6')-Ig-related sequences. An analysis of three susceptible A. haemolyticus strains indicated that aminoglycoside resistance was abolished by the following three distinct mechanisms: (i) a point mutation in aac(6')-Ig that led to a Met56-->Arg substitution, which was shown by analysis of a revertant to be responsible for the loss of resistance; (ii) a polythymine insertion that altered the reading frame; and (iii) insertion of IS17, a new member of the IS903 family. These observations indicated that AAC(6')-Ig is not essential for the viability of A. haemolyticus, although the aac(6')-Ig gene was detected in all members of this species.     

Overexpression and characterization of the chromosomal aminoglycoside 6'-N-acetyltransferase from Enterococcus faecium

The chromosomal gene aac(6')-Ii, encoding an aminoglycoside 6'-N-acetyltransferase in Enterococcus faecium, renders this organism resistant to moderate levels of many aminoglycoside antibiotics. The ubiquitous presence of aac(6')-Ii in E. faecium complicates the selection of antibiotics for treatment of infections caused by this organism. In view of the importance of this enzyme, we have initiated studies to gain an understanding of its molecular mechanism of acetyl transfer. The AAC(6')-Ii enzyme was overexpressed in Escherichia coli and purified in a simple three-step procedure which yields 55 mg of pure dimeric protein per liter of cell culture. Steady-state kinetic analyses revealed a broad substrate specificity and demonstrated that acetylation occurs exclusively at position N-6'. k(cat)/Km values were on the order of 10(4) M(-1) s(-1), which is relatively low compared to other aminoglycoside-modifying enzymes. In addition, MIC values were positively correlated with k(cat), the rate when the enzyme is saturated with the aminoglycoside substrate, and not with k(cat)/Km, the rate at low aminoglycoside (sub-Km) concentrations. These results describe an enzyme which is not optimally evolved for aminoglycoside inactivation and suggest that this chromosomally encoded enzyme may have an alternate physiological function.     

An extracellular factor regulating expression of the chromosomal aminoglycoside 2'-N-acetyltransferase of Providencia stuartii

The chromosomal aac(2')-Ia gene in Providencia stuartii encodes a housekeeping 2'-N-acetyltransferase [AAC(2')-Ia] involved in the acetylation of peptidoglycan. In addition, the AAC(2')-Ia enzyme also acetylates and confers resistance to the clinically important aminoglycoside antibiotics gentamicin, tobramycin, and netilmicin. Expression of the aac(2')-Ia gene was found to be strongly influenced by cell density, with a sharp decrease in aac(2')-Ia mRNA accumulation as cells approached stationary phase. This decrease was mediated by the accumulation of an extracellular factor, designated AR (for acetyltransferase repressing)-factor. AR-factor was produced in both minimal and rich media and acted in a manner that was strongly dose dependent. The activity of AR-factor was also pH dependent, with optimal activity at pH 8.0 and above. Biochemical characterization of conditioned media from P. stuartii has shown that AR-factor is between 500 and 1,000 Da in molecular size and is heat stable. In addition, AR-factor was inactivated by a variety of proteases, suggesting that it may be a small peptide.     

Spread of amikacin resistance in Acinetobacter baumannii strains isolated in Spain due to an epidemic strain

Sixteen amikacin-resistant clinical Acinetobacter baumannii isolates from nine different hospitals in Spain were investigated to determine whether the high incidence of amikacin-resistant A. baumannii was due to the dissemination of an amikacin-resistant strain or to the spread of an amikacin resistance gene. The epidemiological relationship studied by repetitive extragenic palindromic PCR and low-frequency restriction analysis of chromosomal DNA showed that the same clone was isolated in eight of nine hospitals, although other clones were also found. The strains were studied for the presence of the aph(3')-VIa and aac(6')-I genes, which encode enzymes which inactivate amikacin, by PCR. All 16 clinical isolates had positive PCRs with primers specific for the amplification of the aph(3')-VIa gene, whereas none had a positive reaction for the amplification of the aac(6')-I gene. Therefore, the high incidence of amikacin resistance among clinical A. baumannii isolates in Spain was mainly due to an epidemic strain, although the spread of the aph(3')-VI gene cannot be ruled out.     

Molecular cloning and nucleotide sequencing of a novel aminoglycoside 6'-N-acetyltransferase gene from an R-plasmid of Salmonella typhimurium S24 isolated in Taiwan

A conjugative aminoglycoside resistance plasmid pST2 has been isolated from Escherichia coli K-12 14R525, which was mated with a clinical isolate of Salmonella typhimurium S24. A novel resistance gene of aminoglycoside 6'-N-acetyltransferase[AAC(6')] was cloned from plasmid pST2 on a 1,393 kilobase (kb) of SphI-SalI fragment into vector pACYC184 and pUC18. This novel AAC(6') gene in plasmid pST2 acetylated kanamycin, amikacin, dibekacin, tobramycin, gentamicin, netilmicin, and sisomicin. The complete nucleotide sequence of the novel AAC(6') gene and its neighboring sequences were also determined. Minicell experiments detected only one protein of 24.7 kilodaltons (kDa) translated from an open reading frame of the 618 base pairs (bp) gene.     

Abnormal regulation of the Ke 6 gene, a new 17beta-hydroxysteroid dehydrogenase in the cpk mouse kidney

The function encoded by the Ke 6 gene has been recently determined to be 17beta-hydroxysteroid dehydrogenase. Previously, the abnormal expression of the Ke 6 gene has been intimately associated with development of recessive polycystic kidney disease. The Ke 6 gene is normally expressed at very high levels in the kidney and liver and is severely down regulated in all recessive murine models of polycystic kidney disease that have been examined to date. Here, we report a detailed examination of the promoter region of the Ke 6 gene in normal mouse kidney cells (CTA) and in cells derived from mouse kidneys homozygous for the cpk (congenital polycystic kidney) mutation, using transfection analysis and DNA-protein gel shift assays. The minimal promoter region, P1 (+1 to -96), and a putative enhancer site, P3 (-165 to -256), within the Ke 6 gene 5' flanking sequence have been identified. We have also identified another region, P2 (-97 to -165), that may be responsible for the lower promoter activity of the Ke 6 gene in cpk cells. Furthermore, absence of binding of a 38 kDa nuclear protein to a 16 bp sequence element (P1A) within the minimal promoter of the Ke 6 gene suggests that the P1A element could be responsible for the overall reduction in promoter function in cpk cells.     

Evolution of the mitochondrial ATPase 6 gene in Drosophila: unusually high level of polymorphism in D. melanogaster

We have determined 1990 bp mitochondrial DNA sequence which extends from 3' end of the cytochrome oxidase subunit I (COI) gene to 5' end of the COIII gene from two sibling species of Drosophila, D. simulans and D. mauritiana. Analyses of the sequences and part of the NADH dehydrogenase subunit 2 gene and the COI gene together with those from D. melanogaster and D. yakuba revealed that amino-acid substitution rate of the ATPase 6 gene seems to be higher in some strains of D. melanogaster than in the other species. High level of amino-acid polymorphism in this gene was observed in D. melanogaster. Synonymous substitution rate is relatively constant in all the genes examined, suggesting that mutation rate is not higher in the ATPase 6 gene of D. melanogaster. The amino-acid substitutions found specifically in D. melanogaster are at the sites which are not conserved among mammals, yeast and E. coli. These sites of the ATPase 6 gene might lose the selective constraint in D. melanogaster, and the amino-acid substitutions can be explained by neutral mutations and random genetic drift.     

Novel OXA-10-derived extended-spectrum beta-lactamases selected in vivo or in vitro

A clinical isolate of Pseudomonas aeruginosa, PAe191, was found to be highly resistant to all anti-Pseudomonas beta-lactam antibiotics (except imipenem) and resistant also to aminoglycosides. It produced a beta-lactamase (with an apparent pI of 7.6) which was not inhibited by clavulanic acid. Cloning and characterization of the beta-lactamase gene showed that it coded for a novel extended-spectrum OXA-10 variant, called OXA-19, which differed from OXA-10 by nine amino acids and from OXA-13 by two, i.e., Asn in position 73 (Asn73) instead of Ser and Asp157 instead of Gly. Asparagine in position 157 is implicated in resistance to ceftazidime, while the amino acid in position 73, in this variant, seems to condition the level of resistance to penicillins. The oxa19 gene was found to be inserted, in a typical integron structure, immediately downstream from an aac(6')-Ib gene coding for an aminoglycoside acetyltransferase variant, which was called AAC(6')-Ib9.     

The effect of novel polymorphisms in the interleukin-6 (IL-6) gene on IL-6 transcription and plasma IL-6 levels, and an association with systemic-onset juvenile chronic arthritis

During active disease, patients with systemic-onset juvenile chronic arthritis (S-JCA) demonstrate a rise and fall in serum interleukin-6 (IL-6) that parallels the classic quotidian fever. To investigate the possibility that this cytokine profile results from a difference in the control of IL-6 expression, we examined the 5' flanking region of the IL-6 gene for polymorphisms. A G/C polymorphism was detected at position -174. In a group of 383 healthy men and women from a general practice in North London, the frequency of the C allele was 0.403 (95% confidence interval 0.37-0.44). In comparison, 92 patients with S-JCA had a different overall genotype frequency, especially those with onset of disease at < 5 yr of age. This was mainly due to the statistically significant lower frequency of the CC genotype in this subgroup. When comparing constructs of the 5' flanking region (-550-+61 bp) in a luciferase reporter vector transiently transfected into HeLa cells, the -174C construct showed 0.624+/-0.15-fold lower expression than the -174G construct. After stimulation with LPS or IL-1, expression from the -174C construct did not significantly change after 24 h, whereas expression from the -174G construct increased by 2.35+/-0.10- and 3.60+/-0.26-fold, respectively, compared with the unstimulated level. Plasma levels of IL-6 were also measured in 102 of the healthy subjects, and the C allele was found to be associated with significantly lower levels of plasma IL-6. These results suggest that there is a genetically determined difference in the degree of the IL-6 response to stressful stimuli between individuals. The reduced frequency of the potentially protective CC genotype in young S-JCA patients may contribute to its pathogenesis. Similarly the individual's IL-6 genotype may be highly relevant in other conditions where IL-6 has been implicated, such as atherosclerosis.     

Four ubiquitously expressed genes, RD (D6S45)-SKI2W (SKIV2L)-DOM3Z-RP1 (D6S60E), are present between complement component genes factor B and C4 in the class III region of the HLA

The association of the HLA class III region with many diseases motivates the investigation of unidentified genes in the 30-kb segment between complement component genes Bf and C4. RD, which codes for a putative RNA binding protein, is 205 bp downstream of Bf. SKI2W (HGMW-approved symbol SKIV2L), a DEVH-box gene probably involved in RNA turnover, is 171 bp downstream of RD (HGMW-approved symbol D6S45). RP1 (HGMW-approved symbol D6S60E) is located 611 bp upstream of C4. The DNA sequence between human RD and RP1 was determined and the exon-intron structure of SKI2W elucidated. SKI2W consists of 28 exons. The putative RNA helicase domain of Ski2w is encoded by 9 exons. Further analysis of the 2.5-kb intergenic sequence between SKI2W and RP1 led to the discovery of DOM3Z. The full-length cDNA sequence of DOM3Z encodes 396 amino acids with a leucine zipper motif. Dom3z-related proteins are present in simple and complex eukaryotes. In Caenorhabditis elegans, Dom3z-related protein could be involved in the development of germ cells. Human RD-SKI2W and DOM3Z-RP1 are arranged as two head-to-head oriented gene pairs with unmethylated CpG sequences at the common 5' regulatory region of each gene pair. The ubiquitous expression pattern suggests that these four genes are probably housekeeping genes.     

Molecular cloning of cDNA encoding the alpha unit of CNGC gene from human fetal heart

Cyclic nucleotide-gated ion channels (CNGCs) play crucial roles in visual and olfactory signal transduction. As a first step to explore the presence of a CNGC gene in human heart, we cloned a human heart CNGC gene. The sequence consists of 111 bp 5' non-coding region and a 2064 bp open reading frame which is followed by a 459 bp 3' non-coding region. The predicted protein consists of 688 amino acids with a short highly charged segment rich in lysine and glutamate. Sequence comparison indicates that the human heart cDNA is almost identical to the retinal rod photo receptor CNGC cDNA. However, the human cardiac cDNA is lacking a 205 bp Alu fragment in the 5'-uncoding region, has a glutamic acid residue at amino acid position 129, and has a replacement of glutamic acid with a lysine residue at amino acid position 99. Data obtained with northern blot analysis confirm the presence of RNA for the CNGC alpha chain. This channel might play a role in cyclic nucleotide-mediated cellular processes, such as the inotropic effect in the heart.     

Advantage of polymerase chain reaction in the diagnosis of herpes simplex encephalitis: presentation of 5 atypical cases

Exon trapping from cosmids mapping to chromosome 19q13.3 yielded 6 exonic sequences that matched the human symplekin gene, which encodes a tight junction-related protein. One exonic sequence identified a 4.0 kb brain cDNA clone, R6E1, which contained 302 bp 5' to the originally reported 3.7 kb symplekin cDNA. A portion of this novel 5' sequence matched an additional trapped exonic sequence which was obtained from the most telomeric cosmid analyzed. The symplekin gene thus lies in a telomeric-to-centromeric direction on 19q13.3. Only three cosmids from a large 19q13.3 contig hybridized with R6E1, thereby assigning the symplekin gene to a 40 kb region immediately telomeric to gene 59 and the DM protein kinase gene. The 5' end of the R6E1 clone has a potential initiation codon with a strong Kozak sequence and Northern blot analysis detected a 4.2 kb signal in most human tissues, indicating that R6E1 may be a complete cDNA sequence. Based on the trapped exonic sequences, twelve exon-intron boundaries were predicted.     

Cloning and DNA sequence analysis of an aac(3)-Vb gene from Serratia marcescens

The AAC(3)-V resistance mechanism is characterized by high-level resistance to the aminoglycosides gentamicin, netilmicin, 2'-N-ethylnetilmicin, and 6'-N-ethylnetilmicin and moderate resistance levels to tobramycin. Serratia marcescens 82041944 contains an AA(3)-V resistance mechanism as determined from aminoglycoside resistance profiles. This strain, however, does not exhibit hybridization with a probe derived from the previously cloned aac(3)-Va gene, (R. Allmansberger, B. Br?u, and W. Piepersberg, Mol. Gen. Genet. 198:514-520, 1985). High-pressure liquid chromatography analysis of the acetylation products of sisomicin carried out by extracts of S. marcescens 82041944 have demonstrated the presence of an AAC(3) enzyme. We have cloned the gene encoding this acetyltransferase and have designated it aac(3)-Vb. Nucleotide sequence comparisons show that the aac(3)-Va and aac(3)-Vb genes are 72% identical. The predicted AAC(3)-Vb protein is 28,782 Da. Comparisons of the deduced amino acid sequences show 75% identity and 84% similarity between the AAC(3)-Va and AAC(3)-Vb proteins. The use of a DNA fragment internal to the aac(3)-Vb as a hybridization probe demonstrated that the aac(3)-Vb gene is very rare in clinical isolates possessing an AAC(3)-V mechanism.