Novel thermostable antibiotic resistance enzymes from the Atlantis II Deep Red Sea brine pool

The advent of metagenomics has greatly facilitated the discovery of enzymes with useful biochemical characteristics for industrial and biomedical applications, from environmental niches. In this study, we used sequence‐based metagenomics to identify two antibiotic resistance enzymes from the secluded, lower convective layer of Atlantis II Deep Red Sea brine pool (68°C, ~2200 m depth and 250‰ salinity). We assembled > 4 000 000 metagenomic reads, producing 43 555 contigs. Open reading frames (ORFs) called from these contigs were aligned to polypeptides from the Comprehensive Antibiotic Resistance Database using BLASTX. Two ORFs were selected for further analysis. The ORFs putatively coded for 3′‐aminoglycoside phosphotransferase [APH(3′)] and a class A beta‐lactamase (ABL). Both genes were cloned, expressed and characterized for activity and thermal stability. Both enzymes were active in vitro, while only APH(3′) was active in vivo. Interestingly, APH(3′) proved to be thermostable (T_m = 61.7°C and ~40% residual activity after 30 min of incubation at 65°C). On the other hand, ABL was not as thermostable, with a T_m = 43.3°C. In conclusion, we have discovered two novel AR enzymes with potential application as thermophilic selection markers.     

Nucleotide sequence analysis of the gene specifying the bifunctional 6''-aminoglycoside acetyltransferase 2"-aminoglycoside phosphotransferase enzyme in Streptococcus faecalis and identification and cloning of gene regions specifying the two activities.

The gene specifying the bifunctional 6'-aminoglycoside acetyltransferase [AAC(6')] 2"-aminoglycoside phosphotransferase [APH(2")] enzyme from the Streptococcus faecalis plasmid pIP800 was cloned in Escherichia coli. A single protein with an apparent molecular weight of 56,000 was specified by this cloned determinant as detected in minicell experiments. Nucleotide sequence analysis revealed the presence of an open reading frame capable of specifying a protein of 479 amino acids and with a molecular weight of 56,850. The deduced amino acid sequence of the bifunctional AAC(6')-APH(2") gene product possessed two regions of homology with other sequenced resistance proteins. The N-terminal region contained a sequence that was homologous to the chloramphenicol acetyltransferase of Bacillus pumilus, and the C-terminal region contained a sequence homologous to the aminoglycoside phosphotransferase of Streptomyces fradiae. Subcloning experiments were performed with the AAC(6')-APH(2") resistance determinant, and it was possible to obtain gene segments independently specifying the acetyltransferase and phosphotransferase activities. These data suggest that the gene specifying the AAC(6')-APH(2") resistance enzyme arose as a result of a gene fusion.     

The detection of diverse aminoglycoside phosphotransferases within natural populations of actinomycetes

The conserved nature of the genes that code for actinomycete secondary metabolite biosynthetic pathways suggests a common evolutionary ancestor and incidences of lateral gene transfer. Resistance genes associated with these biosynthetic pathways also display a high degree of similarity. Actinomycete aminoglycoside phosphotransferase antibiotic resistance enzymes (APH) are coded for by such genes and are therefore good targets for evaluating the bioactive potential of actinomycetes. A set of universal PCR primers for APH encoding genes was used to probe genomic DNA from three collections of actinomycetes to determine the utility of molecular screening. An additional monitoring of populations for the predominance of specific classes of enzymes to predict the potential of environmental sites for providing isolates with interesting metabolic profiles. Approximately one-fifth of all isolates screened gave a positive result by PCR. The PCR products obtained were sequenced and compared to existing APH family members. Sequence analysis resolved the family into nine groups of which six had recognizable phenotypes: 6′-phosphotransferase (APH(6)), 3′-phosphotransferase (APH(3)), hydroxyurea phosphotransferase (HUR), peptide phosphotransferase, hygromycin B phosphotransferase (APH(7″)) and oxidoreductase. The actinomycetes screened fell into seven groups, including three novel groups with unknown phenotypes. The strains clustered according to the environmental site from where they were obtained, providing evidence for the movement of these genes within populations. The value of this as a method for obtaining novel compounds and the significance to the ecology of antibiotic biosynthesis are discussed. Journal of Industrial Microbiology & Biotechnology (2002) 29, 60–69 doi:10.1038/sj.jim.7000260 Received 25 June 2001/ Accepted in revised form 26 March 2002     

Chimeric genes as dominant selectable markers in plant cells

Opine synthases are enzymes produced in dicotyledonous plants as the result of a natural gene transfer phenomenon. Agrobacteria contain Ti plasmids that direct the transfer, stable integration and expression of a number of genes in plants, including the genes coding for octopine or nopaline synthase. This fact was used as the basis for the construction of a number of chimeric genes combining the 5' upstream promoter sequences and most of the untranslated leader sequence of the nopaline synthase (nos) gene with the coding sequence of two bacterial genes: the aminoglycoside phosphotransferase (APH(3')II) gene of Tn5 and the methotrexate-insensitive dihydrofolate reductase (DHFR Mtx^R) of the R67 plasmid. The APH(3')II enzyme inactivates a number of aminoglycoside antibiotics such as kanamycin, neomycin and G418. Kanamycin, G418 and methotrexate are very toxic to plants. The chimeric NOS-APH(3')II gene, when transferred to tobacco cells using the Ti plasmid as a gene vector, was expressed and conferred resistance to kanamycin to the plant cells. Kanamycin-resistant tobacco cells were shown to contain a typical APH(3')II phosphorylase activity. This chimeric gene can be used as a potent dominant selectable marker in plants. Similar results were also obtained with a NOS-DHFR Mtx^R gene. Our results demonstrate that foreign genes are not only transferred but are also functionally expressed when the appropriate constructions are made using promoters known to be active in plant cells.     

Structure and function of APH(4)-Ia, a hygromycin B resistance enzyme

The aminoglycoside phosphotransferase (APH) APH(4)-Ia is one of two enzymes responsible for bacterial resistance to the atypical aminoglycoside antibiotic hygromycin B (hygB). The crystal structure of APH(4)-Ia enzyme was solved in complex with hygB at 1.95 Å resolution. The APH(4)-Ia structure adapts a general two-lobe architecture shared by other APH enzymes and eukaryotic kinases, with the active site located at the interdomain cavity. The enzyme forms an extended hydrogen bond network with hygB primarily through polar and acidic side chain groups. Individual alanine substitutions of seven residues involved in hygB binding did not have significant effect on APH(4)-Ia enzymatic activity, indicating that the binding affinity is spread across a distributed network. hygB appeared as the only substrate recognized by APH(4)-Ia among the panel of 14 aminoglycoside compounds. Analysis of the active site architecture and the interaction with the hygB molecule demonstrated several unique features supporting such restricted substrate specificity. Primarily the APH(4)-Ia substrate-binding site contains a cluster of hydrophobic residues that provides a complementary surface to the twisted structure of the substrate. Similar to APH(2″) enzymes, the APH(4)-Ia is able to utilize either ATP or GTP for phosphoryl transfer. The defined structural features of APH(4)-Ia interactions with hygB and the promiscuity in regard to ATP or GTP binding could be exploited for the design of novel aminoglycoside antibiotics or inhibitors of this enzyme.     

The aacA-aphD gentamicin and kanamycin resistance determinant of Tn4001 from Staphylococcus aureus: expression and nucleotide sequence analysis

The aacA-aphD aminoglycoside resistance determinant of the Staphylococcus aureus transposon Tn4001, which specifies resistance to gentamicin, tobramycin and kanamycin, has been cloned and shown to express these resistances in Escherichia coli. The determinant encoded a single protein with an apparent size of 59 kDa which specified both aminoglycoside acetyltransferase [AAC(6')] and aminoglycoside phosphotransferase [APH(2")] activities. Nucleotide sequence analysis of the determinant showed it to be capable of encoding a 479-amino-acid protein of 56.9 kDa. analysis of Tn1725 insertion mutants of the determinant indicated that resistance to tobramycin and kanamycin is due to the AAC activity specified by, approximately, the first 170 amino acids of the predicted protein sequence and is consistent with the gentamicin resistance, specified by the APH activity, being encoded within the C-terminal region of the protein. Comparison of the C-terminal end of the predicted amino acid sequence with the reported sequences of 13 APHs and a viomycin phosphotransferase revealed a region which is highly conserved among these phosphotransferases.     

Gene Cassette PCR: Sequence-Independent Recovery of Entire Genes from Environmental DNA

The vast majority of bacteria in the environment have yet to be cultured. Consequently, a major proportion of both genetic diversity within known gene families and an unknown number of novel gene families reside in these uncultured organisms. Isolation of these genes is limited by lack of sequence information. Where such sequence data exist, PCR directed at conserved sequence motifs recovers only partial genes. Here we outline a strategy for recovering complete open reading frames from environmental DNA samples. PCR assays were designed to target the 59-base element family of recombination sites that flank gene cassettes associated with integrons. Using such assays, diverse gene cassettes could be amplified from the vast majority of environmental DNA samples tested. These gene cassettes contained complete open reading frames, the majority of which were associated with ribosome binding sites. Novel genes with clear homologies to phosphotransferase, DNA glycosylase, methyl transferase, and thiotransferase genes were identified. However, the majority of amplified gene cassettes contained open reading frames with no identifiable homologues in databases. Accumulation analysis of the gene cassettes amplified from soil samples showed no signs of saturation, and soil samples taken at 1-m intervals along transects demonstrated different amplification profiles. Taken together, the genetic novelty, steep accumulation curves, and spatial heterogeneity of genes recovered show that this method taps into a vast pool of unexploited genetic diversity. The success of this approach indicates that mobile gene cassettes and, by inference, integrons are widespread in natural environments and are likely to contribute significantly to bacterial diversity.     

Gene cassette PCR: sequence-independent recovery of entire genes from environmental DNA.

The vast majority of bacteria in the environment have yet to be cultured. Consequently, a major proportion of both genetic diversity within known gene families and an unknown number of novel gene families reside in these uncultured organisms. Isolation of these genes is limited by lack of sequence information. Where such sequence data exist, PCR directed at conserved sequence motifs recovers only partial genes. Here we outline a strategy for recovering complete open reading frames from environmental DNA samples. PCR assays were designed to target the 59-base element family of recombination sites that flank gene cassettes associated with integrons. Using such assays, diverse gene cassettes could be amplified from the vast majority of environmental DNA samples tested. These gene cassettes contained complete open reading frames, the majority of which were associated with ribosome binding sites. Novel genes with clear homologies to phosphotransferase, DNA glycosylase, methyl transferase, and thiotransferase genes were identified. However, the majority of amplified gene cassettes contained open reading frames with no identifiable homologues in databases. Accumulation analysis of the gene cassettes amplified from soil samples showed no signs of saturation, and soil samples taken at 1-m intervals along transects demonstrated different amplification profiles. Taken together, the genetic novelty, steep accumulation curves, and spatial heterogeneity of genes recovered show that this method taps into a vast pool of unexploited genetic diversity. The success of this approach indicates that mobile gene cassettes and, by inference, integrons are widespread in natural environments and are likely to contribute significantly to bacterial diversity.     

Crystal structure and kinetic mechanism of aminoglycoside phosphotransferase-2��-IVa

Acquired resistance to aminoglycoside antibiotics primarily results from deactivation by three families of aminoglycoside-modifying enzymes. Here, we report the kinetic mechanism and structure of the aminoglycoside phosphotransferase 2″-IVa (APH(2″)-IVa), an enzyme responsible for resistance to aminoglycoside antibiotics in clinical enterococcal and staphylococcal isolates. The enzyme operates via a Bi-Bi sequential mechanism in which the two substrates (ATP or GTP and an aminoglycoside) bind in a random manner. The APH(2″)-IVa enzyme phosphorylates various 4,6-disubstituted aminoglycoside antibiotics with catalytic efficiencies (k_cat/K_m) of 1.5 × 10³ to 1.2 × 10⁶ (M⁻¹ s⁻¹). The enzyme uses both ATP and GTP as the phosphate source, an extremely rare occurrence in the phosphotransferase and protein kinase enzymes. Based on an analysis of the APH(2″)-IVa structure, two overlapping binding templates specifically tuned for hydrogen bonding to either ATP or GTP have been identified and described. A detailed understanding of the structure and mechanism of the GTP-utilizing phosphotransferases is crucial for the development of either novel aminoglycosides or, more importantly, GTP-based enzyme inhibitors which would not be expected to interfere with crucial ATP-dependent enzymes.     

Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5

The nucleotide sequence of 1200 bp from the unique region of transposon Tn5 containing the neomycin phosphotransferase gene (neo) was determined, and the location of the neo gene was identified by deletion mutants in a translational reading frame of 792 bp. The derived gene product, an aminoglycoside 3′-phosphotransferase (APH) II, consists of 264 amino acid residues and has a calculated M_r of 29053. Its amino acid sequence shows sequence homologies to the APH type I enzyme coded for by transposon Tn903 (Oka et al., 1981).     

The crystal structure of aminoglycoside-3'-phosphotransferase-IIa,an enzyme responsible for antibiotic resistance

A major factor in the emergence of antibiotic resistance is the existence of enzymes that chemically modify common antibiotics. The genes for these enzymes are commonly carried on mobile genetic elements, facilitating their spread. One such class of enzymes is the aminoglycoside phosphotransferase (APH) family, which uses ATP-mediated phosphate transfer to chemically modify and inactivate aminoglycoside antibiotics such as streptomycin and kanamycin. As part of a program to define the molecular basis for aminoglycoside recognition and inactivation by such enzymes, we have determined the high resolution (2.1A) crystal structure of aminoglycoside-3'-phosphotransferase-IIa (APH(3')-IIa) in complex with kanamycin. The structure was solved by molecular replacement using multiple models derived from the related aminoglycoside-3'-phosphotransferase-III enzyme (APH(3')-III), and refined to an R factor of 0.206 (R(free) 0.238). The bound kanamycin molecule is very well defined and occupies a highly negatively charged cleft formed by the C-terminal domain of the enzyme. Adjacent to this is the binding site for ATP, which can be modeled on the basis of nucleotide complexes of APH(3')-III; only one change is apparent with a loop, residues 28-34, in a position where it could fold over an incoming nucleotide. The three rings of the kanamycin occupy distinct sub-pockets in which a highly acidic loop, residues 151-166, and the C-terminal residues 260-264 play important parts in recognition. The A ring, the site of phosphoryl transfer, is adjacent to the catalytic base Asp190. These results give new information on the basis of aminoglycoside recognition, and on the relationship between this phosphotransferase family and the protein kinases.     

Evidence for the location of foreign genes in three different chromosomes in a plant obtained from direct DNA transfer

Tobacco mesophyll protoplasts were treated with plasmids, pCT2 (17.1 kbp) or pCT2T3 (18.3 kbp), which contained a chimeric aminoglycoside phosphotransferase II (APH(3′)II) gene and an intact nopaline synthase gene. Expression of two marker enzymes, APH(3′)II and nopaline synthase, were analyzed in transformed plants. Four out of 16 transformants obtained by pCT2T3 possessed both enzymes. Upon self-pollination, the progeny of one of transformants (T2) segregated to 153∶4 in terms of resistant and susceptible character to kanamycin, suggesting insertion of foreign genes into three independent chromosomes. The kanamycin resistant character in the rest of transformants showed 3∶1 segregation. DNA blot analysis of the T2 transformant and progenies indicated the presence of two marker genes.     

Comparative study of apramycin-resistant microorganisms isolated from man and animals

Apramycin-modifying strains isolated from pigs with coli bacteriosis, from humans and hospital environment were studied comparatively. Production of enzymes modifying the aminoglycoside was estimated with the radioactive cofactor procedure. E. coli isolates from the animals were phenotypically resistant to apramycin and a number of other aminoglycosides. They produced acetyltransferase AAC(3)IV, phosphotransferase APH(3')(5"), APH(3") and other enzymes. Resistance of the strains to gentamicin was also conditioned by AAC(3)IV since these strains did not produce AAD(2") and AAC(6'). In the resistant strains of E. coli and their transconjugates there were detected plasmids with a relative molecular weight of 60-80 MD. Some of the belonged to the compatibility group I1, the others belonged to the compatibility group H1. Strains of S. marcescens, K. pneumoniae. K. oxytoca and S. aureus isolated from humans and hospital environment were sensitive to apramycin. Only isolates of P. aeruginosa were resistant to this antibiotic. However, all the isolates produced AAC(3)IV. Some of them additionally produced AAC(6'), an enzyme modifying amikacin, kanamycin and other antibiotics and not acetylating apramycin. Almost all the strains produced kanamycin- and streptomycin phosphotransferases. Possible coselection of strains resistant to apramycin and gentamicin using one of these aminoglycosides is discussed.     

Promoters active in mammalian cells (pSV40) and in plants (pNOS) permit expression in yeast of the Tn5 APH(3') II gene

Abstract Four plasmids were constructed by associating Escherichia coli and yeast selection markers and replication origins to a structural gene coding for aminoglycoside phosphotransferase (APH(3')) controlled by different flanking sequences. We used the two bacterial genes of Tn5 (APH(3')II) and Tn903 (APH(3')I) as such and the chimeric pSVneo (APH(3')II) and pNOSneo (APH(3')II) constructs, functional in mammalian and plant cells, respectively. Yeast clones resistant to G418 were obtained with all plasmids except with that bearing the bacterial APH(3')II gene. The three plasmids harbouring the functional APH genes, however, conferred different levels of G418 resistance to yeast.     

Intracellular kinase inhibitors selected from combinatorial libraries of designed ankyrin repeat proteins

The specific intracellular inhibition of protein activity at the protein level allows the determination of protein function in the cellular context. We demonstrate here the use of designed ankyrin repeat proteins as tailor-made intracellular kinase inhibitors. The target was aminoglycoside phosphotransferase (3')-IIIa (APH), which mediates resistance to aminoglycoside antibiotics in pathogenic bacteria and shares structural homology with eukaryotic protein kinases. Combining a selection and screening approach, we isolated 198 potential APH inhibitors from highly diverse combinatorial libraries of designed ankyrin repeat proteins. A detailed analysis of several inhibitors revealed that they bind APH with high specificity and with affinities down to the subnanomolar range. In vitro, the most potent inhibitors showed complete enzyme inhibition, and in vivo, a phenotype comparable with the gene knockout was observed, fully restoring antibiotic sensitivity in resistant bacteria. These results underline the great potential of designed ankyrin repeat proteins for modulation of intracellular protein function.     

Structural basis for dual nucleotide selectivity of aminoglycoside 2'-phosphotransferase IVa provides insight on determinants of nucleotide specificity of aminoglycoside kinases

Enzymatic phosphorylation through a family of enzymes called aminoglycoside O-phosphotransferases (APHs) is a major mechanism by which bacteria confer resistance to aminoglycoside antibiotics. Members of the APH(2″) subfamily are of particular clinical interest because of their prevalence in pathogenic strains and their broad substrate spectra. APH(2″) enzymes display differential preferences between ATP or GTP as the phosphate donor, with aminoglycoside 2″-phosphotransferase IVa (APH(2″)-IVa) being a member that utilizes both nucleotides at comparable efficiencies. We report here four crystal structures of APH(2″)-IVa, two of the wild type enzyme and two of single amino acid mutants, each in complex with either adenosine or guanosine. Together, these structures afford a detailed look at the nucleoside-binding site architecture for this enzyme and reveal key elements that confer dual nucleotide specificity, including a solvent network in the interior of the nucleoside-binding pocket and the conformation of an interdomain linker loop. Steady state kinetic studies, as well as sequence and structural comparisons with members of the APH(2″) subfamily and other aminoglycoside kinases, rationalize the different substrate preferences for these enzymes. Finally, despite poor overall sequence similarity and structural homology, analysis of the nucleoside-binding pocket of APH(2″)-IVa shows a striking resemblance to that of eukaryotic casein kinase 2 (CK2), which also exhibits dual nucleotide specificity. These results, in complement with the multitude of existing inhibitors against CK2, can serve as a structural basis for the design of nucleotide-competitive inhibitors against clinically relevant APH enzymes.     

Structural analyses of nucleotide binding to an aminoglycoside phosphotransferase

3',5"-Aminoglycoside phosphotransferase type IIIa [APH(3')-IIIa] is a bacterial enzyme that confers resistance to a range of aminoglycoside antibiotics while exhibiting striking homology to eukaryotic protein kinases (ePK). The structures of APH(3')-IIIa in its apoenzyme form and in complex with the nonhydrolyzable ATP analogue AMPPNP were determined to 3.2 and 2.4 A resolution, respectively. Furthermore, refinement of the previously determined ADP complex was completed. The structure of the apoenzyme revealed alternate positioning of a flexible loop (analogous to the P-loop of ePK's), occupying part of the nucleotide-binding pocket of the enzyme. Despite structural similarity to protein kinases, there was no evidence of domain movement associated with nucleotide binding. This rigidity is due to the presence of more extensive interlobe interactions in the APH(3')-IIIa structure than in the ePK's. Differences between the ADP and AMPPNP complexes are confined to the area of the nucleotide-binding pocket. The position of conserved active site residues and magnesium ions remains unchanged, but there are differences in metal coordination between the two nucleotide complexes. Comparison of the di/triphosphate binding site of APH(3')-IIIa with that of ePK's suggests that the reaction mechanism of APH(3")-IIIa and related aminoglycoside kinases will closely resemble that of eukaryotic protein kinases. However, the orientation of the adenine ring in the binding pocket differs between APH(3')-IIIa and the ePK's by a rotation of approximately 40 degrees. This alternate binding mode is likely a conserved feature among aminoglycoside kinases and could be exploited for the structure-based drug design of compounds to combat antibiotic resistance.     

Nucleotide sequence of the kanamycin resistance determinant of plasmid RP4: Homology to other aminoglycoside 3′-phosphotransferases

The kanamycin resistance determinant of the broad-host-range plasmid RP4 encodes an aminoglycoside 3'-phosphotransferase of type I. The nucleotide sequence of the kanamycin resistance gene (Kmr) and the right end of the insertion element IS8 of plasmid RP4 has been determined. The gene (816 bp) is located between IS8 and the region (Tra 1) encoding plasmid factors mediating bacterial conjugation. Kmr and Tra 1 are transcribed toward each other. The nucleotide sequence has been compared to five related aphA genes originating from gram-negative and gram-positive organisms and from antibiotic producers. Among these that of Tn903 shares the highest degree of similarity (60%) with the RP4 gene. Significant similarities were also detected between the amino acid sequences of the six enzymes. The C-terminal domains of six different aminoglycoside 3'-phosphotransferases (APH(3'] are highly conserved. They are substantially similar to segments of a variety of enzymes using ATP as cofactor. The role of the C-terminal sequences of APH(3') as potential domains for ATP recognition and binding is discussed.     

Kinetic mechanism of enterococcal aminoglycoside phosphotransferase 2"-Ib

The major mechanism of resistance to aminoglycosides in clinical bacterial isolates is the covalent modification of these antibiotics by enzymes produced by the bacteria. Aminoglycoside 2'-Ib phosphotransferase [APH(2')-Ib] produces resistance to several clinically important aminoglycosides in both Gram-positive and Gram-negative bacteria. Nuclear magnetic resonance analysis of the product of kanamycin A phosphorylation revealed that modification occurs at the 2'-hydroxyl of the aminoglycoside. APH(2')-Ib phosphorylates 4,6-disubstituted aminoglycosides with kcat/Km values of 10(5)-10(7) M-1 s-1, while 4,5-disubstituted antibiotics are not substrates for the enzyme. Initial velocity studies demonstrate that APH(2')-Ib operates by a sequential mechanism. Product and dead-end inhibition patterns indicate that binding of aminoglycoside antibiotic and ATP occurs in a random manner. These data, together with the results of solvent isotope and viscosity effect studies, demonstrate that APH(2')-Ib follows the random Bi-Bi kinetic mechanism and substrate binding and/or product release could limit the rate of reaction.