Aminoglycoside-modifying enzymes

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Aminoglycoside-modifying enzymes Gerard D Wright Bacterial resistance to the aminoglycoside antibiotics is most frequently associated with the expression of modifying enzymes that can phosphorylate, adenylate or acetylate these compounds. The recent availability of representative crystal structures for all three classes of modifying enzymes has greatly expanded our knowledge of enzyme function, and has revealed unexpected and exciting connections to other families of enzymes. Furthermore, the complete genome sequences for several bacteria have revealed many potential aminoglycosideresistance elements. Addresses Antimicrobial Research Centre, Department of Biochemistry, McMaster University, 1200 Main Street West, Hamilton, ON L8N 3Z5, Canada; e-mail: [email protected] Current Opinion in Microbiology 1999, 2:499–503 1369-5274/99/$ — see front matter © 1999 Elsevier Science Ltd. All rights reserved. Abbreviations 3D three-dimensional AAC aminoglycoside N-acetyltransferase ANT aminoglycoside O-adenyltransferase APH aminoglycoside O-phosphotransferase

Introduction Antibiotic resistance is a growing problem for all classes of anti-infective agents. One of the first groups of antibiotics to encounter the challenge of resistance was the aminoglycoside–aminocyclitol family. Initially, the resistance that emerged in organisms such as Mycobacterium tuberculosis was restricted to modification of the antibiotic targets, which we now know to be the bacterial ribosomal rRNA and proteins [1]. As new aminoglycosides came to the clinic, however, the prevalence of chemical modification mechanisms of resistance became dominant. Enzymatic modification of aminoglycosides through kinases (O-phosphotransferases, APHs), O-adenyltransferases (ANTs) and N-acetyltransferases (AACs) has emerged in virtually all clinically relevant bacteria of both Gram-positive and Gram-negative origin [2–4]. The challenge to ensure the continued use of these highly potent antibacterial agents will require the effective management of resistance at several levels. One potential mechanism of circumventing resistance is the development of inhibitors of modification enzymes, a methodology that is now well established in the β-lactam field [5]. This approach requires knowledge of resistance at the molecular and atomic levels for the rational design of inhibitory molecules. The recent availability of 3-dimensional (3D) structures of four aminoglycoside-modifying enzymes covering all three classes has now made this strategy viable. Recent research in these areas is described in this review.

Aminoglycoside phosphotransferases APHs enzymes catalyze the ATP-dependent phosphorylation of specific aminoglycoside hydroxyl groups. There are several classes of these enzymes which have been classified primarily on the basis of substrate specificity. The largest family of APHs are those that catalyze the modification of kanamycin at the 3′-hydroxyl, and much of the research in this area has focused on these enzymes. The crystal structure of APH(3′)-IIIa has been determined to 2.2 Å resolution bound with ADP [6]. The structure revealed a dramatic similarity to protein kinases, despite the very low (<5%) amino acid sequence homology (Figure 1). Since most APHs share significant amino acid sequence homology (>25%), it is virtually assured that the general 3D-structure will be conserved throughout this family of resistance enzymes. Though no doubt the molecular details of substrate recognition will be unique to each enzyme, commensurate with their broad and varied specificities. The relationship of APHs to protein kinases has been further strengthened by the recent report that aminoglycoside kinases can in fact act as serine protein kinases [7•]. A variety of peptide and protein substrates were shown to be phosphorylated at low but significant rates by APH(3′)-IIIa and the kinase activity of the bifunctional enzyme AAC(6′)APH(2′′). Furthermore, the mechanism of phosphoryl transfer between the γ-phosphate of ATP and the hydroxyl group of the aminoglycoside is likely to share significant attributes with the phosphoryl transfer mechanism of protein kinases, and initial research has supported this hypothesis [8]. The recent insights into the phosphoryl transfer mechanism in the protein tyrosine kinase, Csk, support a Figure 1

Three-dimensional structure of APH(3′)-IIIa and mouse cAMPdependent protein kinase (cAPK).

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Table 1 Chromosomal aminoglycoside resistance elements from non-aminoglycoside producing bacteria. Gene or locus

GenBank accession No.

Organism

Function

Reference

aac(6′)-Ii

L12710

Enterococcus faecium

Aminoglycoside 6′-N-acetyltransferase

[17]

aac(6′)-Ic

L06160

Serratia marcescens

Aminoglycoside 6′-N-acetyltransferase

(a)

Rv1347c

Z75555

Mycobacterium tuberculosis

Putative aminoglycoside 6′-N-acetyltransferase

[35]

aac(2′)-Ia

L06156

Providencia stuartii

Aminoglycoside 2′-N-acetyltransferase

[36]

aac(2′)-Ib-d

U41471 U72714 U72743

Mycobacterium fortuitum M. tuberculosis Mycobacterium smegmatis

Aminoglycoside 2′-N-acetyltransferases

[37] [38] [38]

aph(3′)-IIb

X90856

Pseudomonas aeruginosa

Aminoglycoside 3′-O-phosphotransferase

[39]

Hph

X74325

Pseudomonas pseudomallei

Hygromycin 4-O-phosphotransferase

[40]

YcbJ

Z99105

Bacillus subtilis

Putative aminoglycoside kinase

[41]

Rv3225c

Z95120

M. tuberculosis

Putative aminoglycoside kinase

[35]

Rv3817

Z97188

M. tuberculosis

Putative aminoglycoside kinase

[35]

aph(9)

U94857

Legionella pneumophila

Spectinomycin 9-phosphotransferase

[42,43]

ant(6)-Ib (aadk)

M26879

B. subtilis

Streptomycin 6-adenyltransferase

[44]

(a)KJ

Shaw, S Gomez-Lus, KW Shannon, personal communication.

dissociative mechanism akin to the SN1 reaction of carbon chemistry [9,10]. These results are, therefore, highly relevant to the aminoglycoside kinase field, and indicate the direction of future mechanistic research in APHs, now reinforced by the availability of protein structure.

Aminoglycoside acetyltransferases AACs are acetylCoA-dependent acyltransferases that primarily modify amino groups (N-acetyltransferases). However, O-acetyltransfer was recently detected using the acetyltransferase domain of the bifunctional enzyme AAC(6′)-APH(2′′) [11•]. Two aminoglycoside acetyltransferase crystal structures have been determined recently [12••,13••]. The first is AAC(3)-Ia from Serratia marcescens, which has been solved in complex with the reaction prodFigure 2

Three-dimensional structure of AAC(3)-Ia and AAC(6′)-Ii.

uct coenzyme A to 2.3 Å resolution (Figure 2) [12••]. The protein is derived from a plasmid-encoded gene that is widespread in Enterobacteriaceae and confers resistance to a variety of aminoglycosides including the clinically important gentamicin Cs. The fold of the enzyme is similar to that of the protein acylating enzymes yeast histone acetyltransferase HAT1 [14] and N-myristoyltransferase [15]. These enzymes are all members of the GCN5-related N-acyltransferase superfamily [16]. The second crystal structure to be determined is AAC(6′)-Ii, which is chromosomally encoded in all Enterococcus faecium [17]. The structure of the acetylCoA-bound enzyme has been determined to 2.7 Å resolution [13••]. The structure of this enzyme shares a similar fold with AAC(3)-Ia and the protein acetyltransferases as discussed above. Previous research on the substrate specificity of the enzyme revealed relatively low specificity constants (kcat/Km) in the steady state. Furthermore, a positive correlation between aminoglycoside minimal inhibitory concentration (MIC) and the maximal velocity of the enzyme (the point at which aminoglycoside is saturating the enzyme’s capacity to modify it) [18]. This observation, along with the chromosomal origin of the gene, suggested that the enzyme might have another function other than aminoglycoside resistance. The structural similarity prompted an investigation into whether the enzyme could acetytate other substrates including peptides such as polyLys and proteins such as histones, and indeed the enzyme has this capacity [13••]. Thus, AAC(6′)-Ii may have another biological function in E. faecium, possibly protein acetylation, although its significance is cryptic as

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insertional inactivation of the gene revealed no obvious phenotype other than the predicted aminoglycoside sensitivity [17].

501

Figure 3

Aminoglycoside nucleotidyltransferases The structure of ANT(4′)-Ia both in the absence of substrates and as the ternary complex with kanamycin and the non-hydrolyzable ATP analogue, AMPCPP, has been determined (Figure 3) [19,20]. These structures show homology in the protein fold to that of rat DNA-polymerase β [21]. Amino acid sequence alignment along with this structural similarity revealed a new family of AMP transferase enzymes [22]. Unlike the APH family, the ANT enzymes show little overall sequence similarity; however, they all share the core polβ signature, which is required for ATP and Mg2+ binding. Thus, the general fold, at least in the nucleotide-binding domain, is most likely shared among these proteins.

Inhibitors of aminoglycoside-modifying enzymes The emerging structural data now has the potential to be exploited in the design of specific inhibitors of enzyme activity. For example, the similarity between APH(3′)-IIIa and protein kinases inspired a study in which known inhibitors of protein kinases were assessed for the capacity to inhibit APHs [23]. Indeed, various inhibitors of serine/threonine and tyrosine kinases (e.g. the isoquinoline sulfonamides and the flavanoids genistein and quercetin) did show mid-µM inhibition of these enzymes, although they did not reverse antibiotic resistance. Nevertheless, this study provides the proof of concept for continued research in this area. In an elegant series of chemical studies, the Mobashery group [24] synthesized aminoglycoside molecules that can act as mechanism-based enzyme inactivators and affinity labels of APHs. Thus, aminoglycosides containing 2′-NO2 were shown to inactivate APH(3′)-Ia and APH(3′)-IIa in an ATP-dependent fashion [24]. A mechanism was proposed whereby APH-catalyzed phosphorylation of the aminoglycoside 3′-hydroxyl precipitated a non-enzymatic dephosphorylation of the product to yield the highly reactive nitroalkene that could react with a nucleophilic group on the APH to form a covalent adduct (Figure 4). More recently, a series of N-bromoacetylated derivatives of neamine were prepared and shown to inactivate APH(3′)-IIa [25] and covalently modify the enzyme at Glu3 and Asp23 [26]. Although the aminoglycoside kinase inactivators discussed above did not reverse antibiotic resistance, a alternative approach, the synthesis of aminoglycoside molecules that are antibiotics but not substrates for modifying enzymes, has been successful over the past 30 years. For example, tobramycin and dibekacin lack the 3′-hydroxyl group, which is the site of APH(3′)-catalyzed phosphorylation of kanamycin class of aminoglycosides, and are competitive inhibitors of APH(3′) [27] and potent antibacterial agents in their own right [28,29]. Unfortunately, these compounds

Three-dimensional structure of ANT(4′)-Ia.

are substrates for other aminoglycoside kinases such as APH(2′′), which is frequently found in Gram-positive organisms as part of a bifunctional enzyme with acetyltransferase activity [11•], and other common aminoglycoside inactivating enzymes such as AAC(6′)s. The Mobashery group [30] has prepared several analogues of kanamycin and neamine that lack NH2 or OH groups in positions which are common sites for AAC modification, but remote to typical kinase targets hydroxyls (e.g. position 6′). Several of these compounds were very poor substrates for APH(3′)-Ia and APH(3′)-IIa, and exhibited antimicrobial activity in Escherichia coli containing these modifying enzymes. These studies demonstrate the capacity to alter the aminoglycoside chemistry in novel ways in order to evade resistance mediated by several classes of enzymes. The approach, however, is not universal as most of these compounds were effectively phosphorylated by APH(3′)-IIIa [31]. Non-aminoglycoside-based inhibition studies in other classes of aminoglycoside-modifying enzymes have not been extensive. Several years ago, however, a screen of natural products designed to discover compounds that could potentiate aminoglycoside action in resistant bacteria revealed the ANT(2′′)-inhibitor 7-hydroxytropoline [32]. This compound was a competitive inhibitor of ATP and was capable of reversing aminoglycoside resistance in a in vitro assay. Thus, there is good evidence that inhibitors of aminoglycoside-modifying enzymes have the potential to reverse

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Figure 4

HO HO O2N

NH2 O Mg2+, ATP O

2-

HO O3P O

NH2 NH2

HO

APH(3')

O R

NH2 O

NH2 O

HO APH(3′)-Nuc :

N O2 O HO

O2N

NH2

H+

NH2

O R

Pi

O

NH2 NH2

HO O R

OH O

R = H or HO

NH2

OH Inactive APH(3′)

Aminoglycoside

Current Opinion in Microbiology

Proposed mechanism of inactivation of APH(3′) by 2′-NO2-containing aminoglycosides. APH(3′) catalyzes the phosphorylation of the 3′-hydroxyl group of aminoglycoside. The aminoglycoside product is

non-enzymatically dephosphorylated to yield a nitroalkene that reacts with the APH(3′) nucleophilic group to form inactive APH(3′).

resistance and rescue antibiotic activity. Although aminoglycoside synthesis or semi-synthesis may result in compounds with both inhibitory and antimicrobial properties, non-aminoglycoside compounds such as the isoquinoline sulfonamide and flavanoid APH inhibitors [23] and the tropolone inhibitors of ANT(2′′) [32] suggest that small molecule inhibitors should also be sought.

the recent availability of representative 3D-structures from the three classes of modifying enzymes: kinases, acetyltransferases and adenyltransferases. The challenge is now to firmly establish the mechanisms of enzyme action and to use this information to prepare effective and potent inhibitors that will reverse antibiotic resistance.

Acknowledgements Prevalence of aminoglycoside-modifying enzymes One of the major challenges of aminoglycoside resistance is the large number and diversity of modifying enzymes. Furthermore, it has been suggested that resistance patterns are influenced by clinical usage of specific aminoglycoside drugs [33]. This is an important finding and it follows that detailed knowledge of the origin and potential reservoirs of resistance would be beneficial in predicting resistance patterns and the continued efficacy of specific drugs. Benveniste and Davies [34] observed over 25 years ago that aminoglycoside-resistance enzymes in clinical isolates and antibiotic-producing organisms were similar, and suggested that these may be a source of resistance elements. The sequencing of many bacterial genes since that time, as well as the growing body of genomic information, has shown that many bacteria have chromosomally encoded putative or bone fide aminoglycoside-resistance genes (Table 1). This indicates that potential aminoglycoside resistance (and in fact other antibiotic resistance) reservoirs are abundant. The clinical implications of this information, however, are not clear at this point. The mechanisms of gene mobilization, and the substrate specificity and catalytic efficiency of these enzymes need to be investigated. However, these results do indicate that resistance elements are probably quite common among bacteria.

Conclusions The understanding of the molecular basis for aminoglycoside-resistance modification has been greatly enhanced by

I thank Albert Berghuis for assistance in preparing Figures 1, 2 and 3. I also thank members of my laboratory and those of my collaborators for their contributions to this field, and the Medical Research Council of Canada for continued financial support.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:

• of special interest •• of outstanding interest 1.

Musser JM: Antimicrobial agent resistance in mycobacteria: molecular genetic insights. Clin Microbiol Rev 1995, 8:496-514.

2.

Wright GD, Berghuis AM, Mobashery S: Aminoglycoside antibiotics: structure, function and resistance. In Resolving the Antibiotic Paradox: Progress in Drug Design and Resistance. Edited by Rosen BP, Mobashery S. New York; Plenum Press; 1998:27-69.

3.

Davies J, Wright GD: Bacterial resistance to aminoglycoside antibiotics. Trends Microbiol 1997, 5:234-240.

4.

Shaw KJ, Rather PN, Hare RS, Miller GH: Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes. Microbiol Rev 1999, 57:138-163.

5.

Sutherland R: Beta-lactam/beta-lactamase inhibitor combinations: development, antibacterial activity and clinical applications. Infection 1995, 23:191-200.

6.

Hon WC, McKay GA, Thompson PR, Sweet RM, Yang DSC, Wright GD, Berghuis AM: Structure of an enzyme required for aminoglycoside resistance reveals homology to eukariotic protein kinases. Cell 1997, 89:887-895.

7. •

Daigle DM, McKay GA, Thompson PR, Wright GD: Aminoglycoside phosphotransferases required for antibiotic resistance are also serine protein kinases. Chem Biol 1998, 6:11-18. This paper demonstrates that the structural similarity between aminoglycoside and protein kinases is extended to similarity in function.

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8.

Thompson PR, Hughes DW, Wright GD: Mechanism of aminoglycoside 3′′-phosphotransferase type IIIa:His188 is not a phosphate-accepting residue. Chem Biol 1996, 3:747-755.

9.

Kim K, Cole PA: Kinetic analysis of a protein tyrosine kinase reaction transition state in the forward and reverse directions. J Am Chem Soc 1998, 120:6851-6858.

10. Kim K, Cole PA: Measurement of a Brønstead nucleophile coefficient and insights into the transition state for a protein tyrosine kinase. J Am Chem Soc 1997, 119:11096-11097. 11. Daigle DM, Hughes DW, Wright GD: Prodigious substrate • specificity of AAC(6′′)-APH(2′′′), an aminoglycoside antibiotic resistance determinant in enterococci and staphylococci. Chem Biol 1999, 6:99-110. This paper provides evidence for aminoglycoside O-acetylation as well as very broad phosphoryl transfer regiospecificity by the bifunctional aminoglycoside-resistance enzyme AAC(6′)-APH(2”). 12. Wolf E, Vassilev A, Makino Y, Sali A, Nakatani Y, Burley SK: Crystal •• structure of a GCN5-related N-acetyltransferase: Serratia marcescens aminoglycoside 3-N-acetyltransferase. Cell 1998, 94:439-449. The authors present the first 3D-structure of aminoglycoside acetyltransferase and demonstrate structural similarity to yeast histone acetyltransferase. 13. Wybenga-Groot L, Draker KA, Wright GD, Berghuis AM: Crystal •• structure of an aminoglycoside 6′′-N-acetyltrasnferase:defiing the GCN5-related N-acetyltransferase superfamily fold. Structure 1999, 7:497-507. This paper describes the 3D-structure of the chromosomally encoded aminoglycoside acetyltransferase (AAC(6′)-Ii). This enzyme is shown to be structural similarity to protein acyltransferases and have protein acetylation capacity. 14. Dutnall RN, Tafrov ST, Sternglanz R, Ramakrishnan V: Structure of the histone acetyltransferase Hat1: a paradigm for the GCN5related N-acetyltransferase superfamily. Cell 1998, 94:427-438. 15. Bhatnagar RS, Futterer K, Farazi TA, Korolev S, Murray CL, JacksonMachelski E, Gokel GW, Gordon JI, Waksman G: Structure of Nmyristoyltransferase with bound myristoylCoA and peptide substrate analogs. Nat Struct Biol 1998, 5:1091-1097. 16. Neuwald AF , Landsman D: GCN5-related histone Nacetyltransferases belong to a diverse superfamily that includes the yeast SPT10 protein. Trends Biochem Sci 1997, 22:154-155. 17.

Costa Y, Galimand M, Leclercq R, Duval J, Courvalin P: Characterization of the chromosomal aac(6′′)-Ii gene specific for Enterococcus faecium. Antimicrob Agents Chemother 1993, 37:1896-1903.

18. Wright GD, Ladak P: Overexpression and characterization of the chromosomal aminoglycoside 6′′-N-acetyltransferase from Enterococcus faecium. Antimicrob Agents Chemother 1997, 41:956-960. 19. Sakon J, Liao HH, Kanikula AM, Benning MM, Rayment I, Holden HM: Molecular structure of kanamycin nucleotidyl transferase determined to 3 Å resolution. Biochemistry 1993, 32:11977-11984. 20. Perdersen LC, Benning MM, Holden HM: Structural investigation of the antibiotic and ATP-binding sites in kanamycin nucleotidyltransferase. Biochemsitry 1995, 34:13305-13311. 21. Holm L, Sander C: DNA polymerase β belongs to an ancient nucleotidyltransferase superfamily. Trends Biol Chem 1995, 20:345-347. 22. Aravind L, Koonin EV: DNA polymerase β-like nucleotidyltransferase superfamily: identification of three new families, classification and evolutionary history. Nucleic Acids Res 1999, 27:1609-1618. 23. Daigle DM, McKay GA, Wright GD: Inhibition of aminoglycoside antibiotic resistance enzymes by protein kinase inhibitors. J Biol Chem 1997, 272:24755-24758. 24. Roestamadji J, Grapsas I, Mobashery S: Mechanism-based inactivation of bacterial aminoglycoside 3′′-phosphotransferases. J Am Chem Soc 1995, 117:80-84. 25. Roestamadji J, Mobashery S: The use of neamine as a molecular template: inactivation of bacterial antibiotic resistance enzyme aminoglycoside 3′′ phosphotransferase type IIa. Bioorg Med Chem Lett 1998, 8:3483-3488. 26. Yang Y, Roestamadji J, Mobashery S, Orlando R: The use of neamine as a molecular template: identification of active site residues in the bacterial antibiotic resistance enzyme

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aminoglycoside 3′′ phosphotransferase type IIa by mass spectroscopy. Bioorg Med Chem Lett 1998, 8:3489-3494. 27.

McKay GA, Wright GD: Kinetic mechanism of aminoglycoside phosphotransferase type IIIa: evidence for a Theorell-Chance mechanism. J Biol Chem 1995, 270:24686-24692.

28. Umezawa S, Tsuchiya T, Muto R, Nishimura Y, Umezawa H: Synthesis of 3′′-deoxykanamycin effective against kanamycin-resistant Escherichia coli and Pseudomonas aeruginosa. J Antibiot 1971, 24:274-275. 29. Umezawa H, Umezawa S, Tsuchiya T, Okazaki Y: 3′′,4′′-Dideoxykanamycin B active against kanamycin-resistant Escherichia coli and Pseudomonas aeruginosa. J Antibiot 1971, 24:485-487. 30. Roestamadji J, Grapsas I, Mobashery S: Loss of individual electrostatic interactions between aminoglycoside antibiotics and resistance enzymes as an effective means to overcoming bacterial drug resistance. J Am Chem Soc 1995, 117:11060-11069. 31. McKay GA, Roestamadji J, Mobashery S, Wright GD: Recognition of aminoglycoside antibiotics by enterococcal-staphylococcal aminoglycoside 3′′-phosphotransferase type IIIa: role of substrate amino groups. Antimicrob Agents Chemother 1996, 40:2648-2650. 32. Allen NE, Alborn WE Jr, Hobbs JN Jr, Kirst HA: 7-Hydroxytropolone: an inhibitor of aminoglycoside-2”-O-adenyltransferase. Antimicrob Agents Chemother 1982, 22:824-831. 33. Miller GH, Sabatelli FJ, Hare RS, Glupczynski Y, Mackey P, Shlaes D, Shimizu K, Shaw KJ, Groups AARS: The most frequent aminoglycoside resistance mechanisms change with time and geographic area: a reflection of aminoglycoside usage patterns? Clin Infect Dis 1997, 24:S46-S62. 34. Benveniste R, Davies J: Aminoglycoside antibiotic-inactivating enzymes in actinomycetes similar to those present in clinical isolates of antibiotic-resistant bacteria. Proc Natl Acad Sci USA 1973, 70:2276-2280. 35. Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D, Gordon SV, Eiglmeier K, Gas S, Barry CE III et al.: Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 1998, 393:537-544. 36. Rather PN, Orosz E, Shaw KJ, Hare R, Miller G: Characterization and transcriptional regulation of the 2′′-N-acetyltransferase gene from Providencia stuartii. J Bacteriol 1993, 175:6492-6498. 37.

Aínsa JA, Martin C, Gicquel B, Gomez-Lus R: Characterization of the chromosomal aminoglycoside 2′′-N-acetyltransferase gene from Mycobacterium fortuitum. Antimicrob Agents Chemother 1996, 40:2350-2355.

38. Aínsa JA, Pérez E, Pelicic V, Berthet FX, Gicquel B, Martín C: 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. Mol Microbiol 1997, 24:431-441. 39. Hachler H, Santarnam P, Kayser FH: Sequence and characterization of a novel chromosomal aminoglycoside phosphotransferase gene aph(3′)-IIb in Pseudomonas aeruginosa. Antimicrob Agents Chemother 1996, 40:1254-1256. 40. Peñaloza-Vazquez A, Mena GL, Herrera-Estrella L, Bailey AM: Cloning and sequencing of genes involved in glyphosphate utilization by Pseudomonas pseudomallei. Appl Environ Microbiol 1995, 161:538-543. 41. Kunst F, Ogasawara N, Moszer I, Albertini AM, Alloni G, Azevedo V, Bertero MG, Bessieres P, Bolotin A, Borchert S et al.: The complete genome sequence of the Gram-positive bacterium Bacillus subtilis. Nature 1997, 390:249-256. 42. Suter TM, Viswanathan VK, Cianciotto NP: Isolation of a gene encoding a novel spectinomycin phosphotransferase from Legionella pneumophila. Antimicrob Agents Chemother 1997, 41:1385-1388. 43. Thompson PR, Hughes DW, Cianciotto NP, Wright GD: Spectinomycin kinase from Legionella pneumophila, characterization of substrate specificity and identification of catalytically important residues. J Biol Chem 1998, 273:14788-14795. 44. Ohmiya K, Tanaka T, Noguchi N, O’Hara K, Kono M: Nucleotide sequence of the chromosomal gene coding for the aminoglycoside 6-adenylyltransferase from Bacillus subtilis Marburg 168. Gene 1989, 78:377-378.