'... .3, .9 V 2‘!” - A}. v“ 2.4.1:} :25 f 1 . ‘2“. “‘ c; .m» 1 M .1. k a ‘ ._. 5:} ‘f 'faé‘cg“. ‘ 9: tiffix’r ‘ ,3; h 151"“; x .Eg. a ‘ : .‘Q‘u hr“, asuk‘fgzg - 1x542: ~~ . 11!»..- . ‘7‘“ ' L -4vM1v v- 5‘ than»; «9". fi . {Km u M” ’2,” flaw ' .2 an. .1- 32> .4‘ “h." "H :I .W ,1 4,7 f‘ . :3: Am: WW finv : ... . 4. -m'»-" o \ ~ . 1 5ng 3 . run..— . V. 3 I- . u ‘ ‘ pd :. ‘M u '- l9. 3:2 ."4’ .... . A- $3? )1 . fidhfij .3??? ' 4: ‘1‘ ev I ,_ ‘. . ‘ q; 4%? ., . . ( o y —:: - 1.199,}. "‘ %~é3‘. , "A: {r 0 31 5r E...” w,"23.-< w, c l THESIS MICHIGAN STATE UNIVERSITY LIBRARIES II III Illllllllllllllllllllllll Hill 3 1293 01022 2606 n I This is to certify that the thesis entitled The Mechanisms of Streptomycin Resistance in Erwinia amylovora presented by Chien-Shun Chiou has been accepted towards fulfillment of the requirements for Ph.D. Botany & Plant Pathology degree in Major of sor Date 12-30-93 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution , 7, ,, _l_ _...__. LIBRARY I Michigan State University PLACE DI RETURN BOX to rornovo this chockout from your rocord. TO AVOID FINES rotum on or botoro onto duo. DATE DUE DATE DUE DATE DUE I_—I I I L—J|__I__I ESE I—T—TI—jl MSU loAnAfiirmotivo Action/EM Opportunity lnotitr‘ion THE MECHANISMS OF STREPTOMYCIN RESISTANCE IN Erwinia amylovora BY Chien-Shun Chiou A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1994 ABSTRACT THE MECHANISMS OF STREPTOMYCIN RESISTANCE IN Erwinia amylovora BY Chien-Shun Chiou The mechanism of streptomycin resistance in Erwinia amylovora, the fire blight pathogen causing the most destructive bacterial disease of apple and pear, has been studied on a molecular genetic and biochemical basis. Streptomycin-resistant mutants of E. amylovora were isolated from an apple orchard in Michigan in 1990. In colony hybridization, a portion of the resistance gene (SMPB) from strain Psp36 of Pseudomonas syringae pv. papulans hybridized with all streptomycin-resistant strains of E. amylovora, but not with streptomycin-sensitive strains. A 34-kb plasmid (pEa34) was present in all streptomycin-resistant field strains but not in streptomycin-sensitive strains. Streptomycin resistance and pEa34 were cotransferred by conjugation into four streptomycin-sensitive recipients. A class II Tn3-type transposable element, designated Tn5393 and located on plasmid pEa34 from streptomycin- resistant strain CA11 of E. amylovora, was identified by its ability to move from pEa34 into recipient plasmid replicons pGEM32f(+) and pUCDBOO. Nucleotide sequence analysis reveals that Tn5393 consists of 6,705 hp with 81-bp terminal inverted repeats and generates 5-bp duplications of the target DNA following insertion. Two open-reading frames, separated by a 194-bp putative recombination site (res), encode a putative transposase (tnpA) and resolvase (tnpR) of 961 and 181 amino acids, respectively. Two streptomycin resistance gene, strA and strB, were identified on the basis of their DNA sequence homology to the resistance genes in plasmid RSF1010. Between tnpR and strA is a 1.2-kb insertion sequence designated 181133. The 3.2-kb tnpA-res- tnpR was detected in P. syringae pv. papulans Psp36 and in many other gram-negative bacteria harboring strA-strB isolated from Michigan apple orchards. Except for some strains of Erwinia herbicola, these other gram-negative bacteria lack insertion sequence 181133. The prevalence of strA-strB could be accounted for by transposition of Tn5393 to conjugative plasmids that are then disseminated widely among gram-negative bacteria. The plasmid-borne strA and strB genes from Erwinia amylovora strain CA11 were characterized by genetic and biochemical analyses. In deletion experiments, deletions in strB resulted in a reduction in the minimum inhibitory concentration (MIC) from 500 to 100 pg streptomycin ml'1 and in strA from 500 to 25 pg streptomycin ml’1 or less. When strA and strB were cloned separately on a lacIQ/Ptac-based expression vector in Escherichia coli, the protein encoded by strA, but not the one by strB, was overexpressed. Sequence analysis of the overlapping genes indicated that the distal strB gene lacked a Shine-Dalgarno sequence and that the initiation codon was in the double-stranded region of the stable stem-loop structure. Conversely, the Shine- Dalgarno sequence and the initiation codon in strA were exposed in the single-stranded loop of a stable stem-loop structure. The strB gene was overexpressed and resistance restored to a MIC of 100 pg streptomycin ml"1 by introducing a Shine-Dalgarno sequence and by altering the mRNA secondary structure. 13C-NMR analysis of the respective phosphorylated streptomycin products indicated that stra- strB encoded aminoglycoside-3"-phosphotransferase [APH(3")- Ib] and aminoglycoside-6-phosphotransferase [APH(6)-Id], respectively. These data suggest that the high level of resistance to streptomycin exhibited by bacteria with stra- strB genes is due to the coexistence in the cells of APH(3")-Ib and APH(6)-Id enzymes and that the differential expression of these enzymes is regulated by the mRNA secondary structures. DEDICATION To my grandparents Shoa Chiou and Jin Chuang, my father Huan Chiou and my mother Juo Chang. They always did their best for their children's education. ii ACKNOWLEDGMENTS I would like to express thanks and appreciation to the persons who have encouraged, guided and assisted me in my Ph.D. journey. To my major professor Dr. Alan L. Jones. He is the most supportive person to my research. His encouragement and patience were extremely helpful to me in completing my degree and recognizing my own capabilities. To the committee members Dr. Shauna Somerville, Dr. Robert Hollingworth, and Dr. Mickil Bagdasarian, whose assistance, support, and thoughtful evaluation are much appreciated. To my lab colleagues Patricia McManus, Lizhe Liang, Harrie Koenraadt, and Gail Ehret for their advice and assistance for my work. Their guidance was especially helpful to my presentations. To my friend Kung-Lung Cheng for his assistance in 13C-NMR work. To my wife, Ishien Li, a brave mother of a daughter and a son. She has to work for a research assistantship, study for her Ph.D. degree and do everything for our children. I also want to thank my brothers for financial and emotional support and to my parents-in-law and brother-in- law for taking care of my children, Janice and Brian Chiou. iii TABLE OF CONTENTS Page LIST OF TABLESoooooooooooooooooooooooooooooooooooooooo Viii LIST OF FIGURES""""""°°"'°°°""'°°"°'°°"°°" ix GENERAL INTRODUCTION AND LITERATURE REVIEW°°°°°°°°°°°° 1 REFERENCESoooooooooooooooooooooooooooooooooooooooooooo 15 PART I THE ANALYSIS OF PLASMID-MEDIATED STREPTOMYCIN RESISTANCE IN Erwinia amylovora ABSTRACToooooooooooooooooooooooooooooooooooooooooooooo 26 INTRODUCTIONoooooooooooo oooooooooo oooooooooo oooooooooo 26 MATERIALS AND METHODS Bacterial strains and plasmids--------°-- -------- 26 Detection of streptomycin-resistant Erwinia amylovoraooooooooooooooooo ..... oooooooooooooooooo 26 Orchard surveyoooooo...oo ...... oooooooooooooooooo 27 Plasmid characterizations------------------------ 27 HYbridizationSooooooooooooooooooooooooooooooooooo 27 Restriction enzyme digests and Southern blots---- 27 Bacterial conjugation.ooooooooooooooooooooooooooo 27 RESULTS Detection of resistance-------------- ..... ....... 27 Level of reSistanceoooooooooooooooooooooooooooooo 27 Distribution of resistant strains-------------°-- 27 Colony hybridization studieso-------------------- 27 Plasmid Characterizationsoooooooooo ..... ooooooooo 27 Southern analysis................. ......... ...... 27 Bacterial conjugation................o........... 28 DISCUSSIONooooooooooooooooooo ooooooo oooooooooooooooooo 28 LITERATURE CITEDoooooooooooooooooooooooo oooooooooooooo 29 iv Page PART II NUCLEOTIDE SEQUENCE ANALYSIS OF A TRANSPOSON (Tn5393) CARRYING STREPTOMYCIN RESISTANCE GENES IN Erwinia amylovora AND OTHER GRAN-NEGATIVE BACTERIA ABSTRACToooooooooooooooooooooooooooooooooooooooooooooo 32 INTRODUCTIONooooooooooooooooooooooooooooo ooooo oooooooo 32 MATERIALS AND METHODS Bacterial strains and plasmids--------------°--" 32 Hybridization studieSoooooooooooooooooooooooooooo 32 Cloning, isolation, and restriction mapping of pEa34oooooooooooooooooooooooooooooooooooooooooooo 32 Detection of a transposon from pEa34----------~-- 33 Cloning and sequencing of DNA'----°------ -------- 33 Detection of Tn5393 and pEa34 in other gram-negative bacteriaooooooooooooooooooooooooooo 33 Nucleotide sequence accession number °°°°°°°°°°°°° . 34 RESULTS Restrictionmap of pEa34ooooooooooooooooooooooooo 34 Detection of a transposable element from pEa34°-- 34 Sequence and coding regions of transposon Tn5393° 34 Identify of streptomycin resistance in P. syringae pv. papulans and E. amylovoranunn 37 Tn5393 in other gram-negative bacteria--°--°--°-° 37 Plasmid pEa34 in other gram-negative bacteria---- 38 DISCUSSIONOOoooooooooooooooooooooooooooooooooooooooooo 38 ACKNOWLEDGMENTSooooooooo oooooo ooooooooo ..... oooo oooooo 39 REFERENCESoooooooo oooooo ooooooooooooooooooooooo ooooooo 39 PART III IDENTIFICATION OF TWO AMINOGLYCOSIDE PHOSPHOTRANSFERASE, APH(3")-Ib AND APH(6)-Id, ENCODED BY THE aph(3")-Ib (strA) AND aph(6)-Id (strB) GENES FROM STREPTOMYCIN-RESISTANT Erwinia amylovora ABSTRACToooooooooooooooooooooooooooooooooooooo oooooooo 42 INTRODUCTIONoooooooooooooooooooooooooooo oooooooooooo oo 44 MATERIALS AND METHODS Bacterial strains, plasmids, and primers--------° 46 DNA manipulations and construction of expression vector pTWNHEoooooooooooooooooooooooooooooooooooo 46 Enzyme assaysoooooooooooooooooooooooooooooooooooo 49 Determination of minimum inhibitory concentration (MIC)"""""""""°"°""“"""""""'° Deletion analysis in the strA and strB region.... Modification of strB and cloning into pTWNHE°°°°° Expression and analysis of strA and strB gene products.oooooooooooooooooooooooooooooooooooooooo N-terminal amino acid sequence determination----- Preparation and 13C-NMR analysis of the phosphorylated streptomycin ooooooooooooooooooooo Secondary structuresoooooooooooooooooooooo ooooooo RESULTS Deletion and complementation analysis of strA and StrBooooooooooooooooooooooooooooooooooooooooooooo Effects of Shine-Dalgarno sequence and mRNA secondary structure on the expression of str --°° Confirmation of strA and strB proteins-------°°-- Identification of the phosphorylated streptomycin productsooooooooooooooooooooooooooooooooooooooooo DISCUSSION°°'°-°°°°°°°°° ......... ............. ........ ACKNOWLEDGMENTS ....................................... REFERENCES°°'°°°°° ........ ............ ..... .... ....... APPENDIX A PROPOSED RESEARCH ON RIBOSOME-MEDIATED STREPTOMYCIN RESISTANCE IN Erwinia amylovora INTRODUCTION................................. ......... OBJECTIVES... ......................... ... ............. JUSTIFICATION"°°°°°°°°°°°°°°°°°°°°°°°'°'°°°°' ........ DESCRIPTION OF RESEARCH PLAN°°°'°°°°'°°'° ......... .... REFERENCES ........................................... APPENDIX B CONJUGATIONAL TRANSFER OF pEa34 CONJUGATIONAL TRANSFER OF pEa34-------------- --------- REFERENCESooooooooooooooooooooooo oooooooo oo ooooooooooo APPENDIX C vi Page 49 50 50 51 52 52 53 54 57 63 63 65 67 68 72 73 74 76 80 84 88 Page IDENTIFICATION OF THE MECHANISMS OF STREPTOMYCIN RESISTANCEQOOQooococoon...oooooooooooooooooooono...on. 90 REFERENCES...00000ooo000.0000ooooooooooooooooooooooooo 97 vii LIST OF TABLES Table Page PART I 1. Frequency of conjugational transfer of resistance from streptomycin-resistant to streptomycin- sensitive Erwinia amylovora----.°°----'°--------°- 28 PART II 1. Bacterial strains and plasmids used in this study- 33 PART III 1. Bacterial strains, plasmids and primerS°-----'-°-- 47 APPENDIX B 1. The transfer frequency of p£a34 by conjugation among strains of Erwinia amylovora, Escherichia coli, Pseudomonas syringae pv. populans and Erwinia herbicolaooooooooooooooooooooooooooooooooo 85 APPENDIX C 1. Characterization of mechanisms of streptomycin resistance by the ability of resistance to streptomycin and myomycin. hybridization to SMP3, and in vitro streptomycin phosphotransferase (APR) and streptomycin adenylytransterase (ANT) assay°-- 96 viii LIST OF FIGURES Figure Page PART I Plasmids in field strains of Erwinia amylovora isolated from apple orchard SW in southwest Michigan and from adjacent crabapple trees. Streptomycin-resistant strains CA13, CA11, HT16, HTOl-l, and HTO6 are in lanes 1, 3, 6, 7. and 8, respectively, and streptomycin-sensitive strains CA10, CA06, HT03, and HTOl are in lanes 2, 4, 9, and 10, respectively. Lane 5 contains Erwinia stewartii SW20oooooooooooooooooooooooooooooooooso. 28 Autoradiograph of a Southern blot of plasmid DNA, first, and total genomic DNA (including plasmid DNA), second, of Pseudomonas syringae pv. papulans strain Psp36 (lanes 1 and 2), and DHl with plasmid pCPPSOS (lanes 3 and 4) and streptomycin-resistant (CA11, lanes 5 and 6; H062-1, lanes 9 and 10) and streptomycin-susceptible (BC06, lanes 7 and 8; ELOl, lanes 11 and 12) strains of Erwinia amylovora digested with AvaI and hybridized to probe SMP3. The sizes (in kb) of hybridizing fragments calculated from EcoRI-HindIII-digested bacteria phage lambda DNA are give at the far left---'--°-- 28 Agarose gel electrophoresis of cleared lysates of donor, recipient, and transconjugant strains of Erwinia amylovora. Lane 1, recipient BC06 rifr; lane 2, transconjugant BC06 rifr X CA11; lane 3, donor CA11; lane 4, transconjugant ELOl rifr X CA11; lane 5, recipient EL01 rifr; lane 6, strain Ea88-9O with plasmid identical in restriction pattern to pEAzg (7)ooooooooooooooooooooooosososo. 28 Ethidium bromide-stained gel (A) and auto- radiograph of a Southern blot (B) of plasmid DNAs of donor, recipient, and transconjugant strains of Erwinia amylovora digested with AvaI and hybridized to probe SMPB. Lane 1, recipient EL01 rifr; lane 2, transconjugant ELOl rifr X CA11; lane 3, donor CA11; lane 4, transconjugant BC06 rifr X CA11; lane 5, recipient BC06 rifr; lane 6, ix Figure Page transconjugant BC06 rifr X H062-1; lane 7 donor H062-1; lane 8, transconjugant EL01 rifr X H062-1; and lane 9, recipient EL01 rifr. The sizes (in kb) of hybridizing fragments calculated from EcoRI-HindIII-digested bacteria phage lambda DNA are given at the far left-00.00.000.00...coo-coo.o 29 PART II (A) Restriction enzyme map of plasmid pEa34 from E. amylovora CA11. Each restriction site is designated on the basis of its distance (in kilobases) from the unique BamHI site. The location of streptomycin resistance genes strA and strB is indicated with a double arrow, and the location of transposon Tn5393 is indicated with a thick line. (B) The proposed genetic map for transposon Tn5393. The proposed elements in Tn5393 are IR, transposase gene (tnpA), region res, resolvase gene (tnpR), insertion element (151133), strA and strB genes, and IR, respectively. The direction of transcription of the genes are indicated with arrows. The locations of probe SMP3 from P. syringae pv. papulans Psp36 and of probe SAC32 are shown by the open boxes immediately below the genetic map. The locations of DNA sequences from RSF1010 and pCPP505 that are identical to the sequence in Tn5393 are shown by the slashed boxes, and the IR sequences are shown by the filed boxes-'------°--° 34 Complete nucleotide sequence of Tn5393, numbered from the IR on the tnpA end, is shown in 5' to 3' orientation. The positions of ORFs corresponding to four predicted polypeptides (A, B, C, and D) are indicated with their encoded amino acid sequence. ORE C is transcribed from the strand opposite to ORFs A, B, and D. The nucleotide sequence of insertion sequence 181133 is from nucleotide 3660 to 4891. The 3-bp direct repeats that border 181133 (TAG) are double underlined, and the IRs of Tn5393 and I81133 are underlined. The -10 and -35 consensus sequences of three putative promoters are indicated above the sequence by a line and the number. Asterisks indicate stop codons. The nucleotide and amino acid sequences of ORF A that differ from those of ORF I in RSF1010 are indicated above the nucleotide and beneath the amino acid, respectively. Sequence 4928 to 6705 of Tn5393 is homologous to sequence 31 to 1808 of RSF1010 (32)° 35 Figure 3. Comparison of IR sequences. (A) The 81-bp IRs of Tn5393 (IR-5393t and IR-5393o, where t is the tnpfl end and o is the other end) and the sequence of plasmid RSF1010. Boldface type shows positions where residues were different. (B) IRs in representative transposons from the Tn3 family (Tn5393, Tn3, Tn21, Tn917, Tn2501, and Tn4430) are all listed from the tnpA end. The stop codons for the transposases in Tn5393, Tn3, Tn21, and Tn2501 are underlined. The number in parentheses is the number of identical base pairs between each IR and IR-5393t. Sequences are from Scholz et al. (32) for RSF1010, Heffron et al. (12) for Tn3, Zheng et al. (45) for Tn21, An and Clewell (1) for Tn917, Michiels and Cornelis (22) for Tn2501, and Mahillon and Lereclus (20) for Tn4430----------°-- Autoradiograph of a Southern blot of plasmid DNAs from streptomycin-resistant (except lane 3) gram- negative bacteria digested with SacI and hybridized to probe SAC32. Lane 1, P. syringae pv. papulans Psp36; lane 2, E. amylovora CA11; lane 3, E. amylovora EL01; lane 4, Acinetobacter sp. strain 1; lane 5, E. herbicola 6a; lane 6, E. herbicola 34; lane 7, a yellow Pseudomonas strain, 40; lane 8, P. syringae pv. syringae 45; lane 9, Pseudomonas fluorescens biovar III, 57; lane 10, Pseudomonas aeruginosa 60; lane 11, P. putida 61; lane 12, Aeromonas sp. strain 145; lane 13, E. herbicola 144. The size of the 3.2-kb hybridizing fragment is indicated at far left°-°---- ---------- Hybridization of a 25. 6-kb SmaI fragment from plasmid pEa34 with AvaI-digested total plasmid DNAs from streptomycin-resistant strains of E. herbicola. Lanes 1 to 6 contain DNAs from strains 52, 50a, 144, 35a, 18, and 6a, respectively. Lane 7 contains plasmid pEa34 from E. amylovora CA11. Strain 6a was a negative control. The three AvaI fragments indicated (by size in kilobases) at the far left are internal fragments of Tn5393. A 127-bp fragment is not seenoooooooooooooooo ooooo PART III (A) Resistance to streptomycin and aminoglycoside phosphotransferase (APH) activity in extracts from cells of Escherichia coli JM109 transformed with various deletions in the strA and strB genes from Erwinia amylovora strain CA11. Deletion derivatives were cloned in vector pGEM3Zf(+) with xi Page 37 38 38 Figure Page the genes oriented downstream from a lac promoter. Bacteria were grown in the presence and absence of isopropyl-B-D-thiogalactopyranoside (IPTG). Bold lines with arrows represent the coding regions of strA and strB and the transcriptional direction for each gene. Numbers above the lines are nucleotide sequence numbers for Tn5393 (4) and "P" indicates a promoter from 181133. (B) Complementation of deletions in strA and str ---°- 56 SDS-PAGE analysis of proteins from strains of Escherichia coli transformed with expression vector pTWNHE with strA, strB, or modified strB variants. Proteins were separated on a 13.5% SDS- polyacrylamide gels and stained with Coomassie blue. Bands labeled 27, 28, and 30 kDa are the protein products from strA, strB, and strB-AJ26N, respectively. (B) The percentage of the expressed proteins from strA and strB variants to the total cellular protein for strains grown in LB medium with (+) or without (- ) IPTG. Data were obtained by scanning four individual lanes for each strain on SDS gels using AMBIS Core Software Version 4.0 (AMBIS, Inc. San Diego, California). (C) Resistance of each strain to streptomycin on LB agar with (+) and without (- ) IPTG°------------°-° 59 Predicted mRNA secondary structures surrounding the ribosome-binding site for strA, strB, and six strB variants. The stem-loop structures and free energy (AG; in kcal mol’l) were determined with the computer program Hairpin Loop Search, DNASIS version 3.00 (Hitachi Software Engineering Co. LTD). Nucleotides in ribosome-binding sites that can pair with the 3' end of E. coli 168 rRNA (AUUCCUCCACU...5') are indicated as larger characters and those that cannot pair with strB sequence are underlined. Initiation codons are enclosed by a box and the stop codon from strA is marked with asterisks. The secondary structures for strB-AJ24 and strB-AJ34 were unaltered, the structures for strB-AJZS, strB-AJ26, strB-AJ27, and strB-AJZB were altered by replacing nucleotides between the Shine-Dalgarno sequence and the initiation codon. Six nucleotides (the first two codons of strB) on strB-AJZ? and strB- AJ28 were omitted. The strain of Escherichia coli transformed with each gene is shown in parenthesis under the gene designationooooooooooooo00000000000 61 xii Figure Page APPENDIX C 1. Molecular structures of streptomycin and spectinomycin and the reaction sites of APH(3"), APH‘G)’ANT(3")' andANT(6)osooooooooooooooooooooo 92 2. Flowchart for identification of streptomycin resistance mechanismsooooooooooooooooooooooo oooooo 94 xiii GENERAL INTRODUCTION AND LITERATURE REVIEW Fire blight is the most devastating bacterial disease of pomaceous fruit trees (106). It is very destructive to pear (Pyrus communis L.) and less so to apple (Malus sylvestris Mill.), quince (Cydonia oblonga Mill.), and several other members of the family Rosaceae. Fire blight may result in tremendous economic losses on pear and apple crops when it occur epidemically. The first reference to fire blight is considered to be a description of a problem on apple trees taken from a letter by W. Denning in 1793. Thereafter, the possible cause of fire blight was debated until 1878 when T. J. Burrill first proposed that bacteria caused the disease. However, the isolation of the bacterium in pure culture and reinoculating it into healthy plants to prove definitely the real causal agent was not completed until 1884 by J. C. Arthur. Fire blight was apparently indigenous to North America. The disease probably occurred on native American plants, such as crab apple, hawthorn, and mountainash. From these native hosts the bacterium probably spread to the susceptible cultivated pears and apples planted by the early American settlers. The occurrence of fire blight was restricted to North America for a century since 1800s. Outside North America, fire blight was first reported in New Zealand in 1919. In Europe the disease was report in England in 1957. The disease has since spread through much of Europe and the Mediterranean area causing serious losses to pome fruits and rosaceous ornamentals. It has not been reported in Asia. The causal organism of fire blight is the bacterium Erwinia amylovora (Burrill 1882) Winslow, Broadhurst, Buchanan, Krumwiede, Rogers, and Smith 1920. E. amylovora, a member of the family Enterobacteriaceae, is a Gram- negative, facultatively anaerobic, peritrichously flagellal, rod-shaped (av. 1.1-1.6 x 0.6-0.9 pm) bacterium. The identification of E. amylovora is usually based on characteristic growth of colonies on one or more differential media (21, 47, 70), immunological tests, the production of bacterial ooze on immature pear fruit following inoculation, and the hypersensitive reaction of Nicotiana tabacum L. to infiltrated bacteria (43, 52, 53, 54). Recently, a specific DNA probe derived from the universal plasmid pEA29 in E. amylovora has been developed for routine, cost-effective identification of this bacterium (32). The methods for the control of fire blight are difficult, expensive, and not very effective. Streptomycin is the most effective bactericide compared to the alternatives, copper- based bactericides, oxytetracycline, and flumequine. Therefore, the emergence of streptomycin-resistant pathogen of fire blight has become a severe problem to the pear and apple industry in North America. Streptomycin is an aminoglycoside-aminocyclitol antibiotic produced by Streptomyces spp. (23, 45). It was first isolated in 1944 by Schatz et a1. (86) and introduced as an antituberculosis drug in 1949. However, it frequently produced serious toxic effects, notably, severe vestibular dysfunction and, occasionally, partial or complete deafness. Perhaps its most serious disadvantage lies in the rapidity with which resistant strains emerge. In plant pathology, streptomycin was mainly introduced to control fire blight. Extensive experimentation on the efficacy of streptomycin for the control of fire blight was conducted in North America in the early 1950s. After streptomycin was registered in the United States in the late 1950s, it was used extensively for the control of fire blight on pears in the West and on apples in the East. Besides fire blight, streptomycin has been used to control bacterial spot of tomato and pepper and blister spot of apple. Outside the United States, streptomycin has also been used in New Zealand and Canada. As early as 1958, M. H. Dye had demonstrated the development of streptomycin resistance in a plant bacterial pathogen in the laboratory (31). In the field, the occurrence, frequency, and distribution of streptomycin resistance in the causal organism of bacterial spot in tomato and pepper, Xanthomonas campestris pv. vesicatoria, were first reported in the early 1960s (98, 104). Resistance to streptomycin has also been reported in several pathovars of Pseudomonas syringae (13, 26, 49, 85, 102, 109) and in strains of X. campestris. pv. dieffenbachiae (55). Streptomycin-resistant E. amylovora was first detected in pear orchards of California in 1971 (70) and then in pear orchards of Washington and Oregon (20, 63). Except for a report of resistant strains on apple in Missouri (91), detection of streptomycin-resistant E. amylovora has until recently been limited to the western United States. In the mid—1970s, attempts to detect resistant strains in apple and pear orchards in western New York State and in apple orchards in Michigan failed (7, 103). A second attempt to detect resistant E. amylovora in 1993 in New York apple growing areas was also unsuccessful (14). In Michigan, the first detection of streptomycin-resistant strains of E. amylovora was in 1990 in an apple orchard in Van Buren County. The characterization of the streptomycin-resistant E. amylovora detected in Michigan in 1990 is the subject of this thesis. Bacteria have versatile ways to cope with antibiotics and other toxic elements (5, 11, 22). The mechanisms of resistance include inactivation of the antibiotics by enzymes, alteration of the antibiotic target, and reduction of drug accumulation (12). Some groups of antibiotics are inactivated by modifying enzymes (22, 93) or destructive enzymes (1, 3). Inactivation of antibiotics by enzymes is the most important and commonest mechanism of resistance since the genes encoding the enzymes are usually carried on self-transmissible or mobilizable plasmids and transposons. Antibiotics effectively inhibit the cellular components and enzymes related to bacterial cell wall synthesis, protein synthesis, and DNA replication (12). It has been found that modification with enzymes and mutations of the target sites may reduce the binding affinity of the target sites to the antibiotics (58, 75, 95, 107). Overproduction of the target protein (41) or production of an insensitive form of the target protein by genes on a plasmid and transposon may also result in resistance (4, 84). Decreased permeability of the plasma membrane to toxic substances or decreased binding of toxic compounds are initially proposed to explain the mechanism of reduced accumulation by resistant cells (48, 57). Recently, active efflux have been proposed to explain the mechanism of reduced drug accumulation (2, 60, 68). In addition to bacteria, active efflux systems, which are responsible for resistance to a variety of structurally unrelated antibiotics and toxic compounds, have been reported in fungi (28), protozoan parasites (56, 66), and mammalian cancer cells (29). In bacteria, active efflux systems may contribute to mechanism of resistance to antibiotic, heavy metals, and other toxic substances (60). Efflux proteins usually contain membrane-spanning units and ATP-binding domains that allow them to serve as energy- dependent transporters, and their genes may occur on plasmids, transposons, or chromosomes. Recent studies have shown that active efflux systems are involved in multidrug resistance in bacteria (19, 77) and in mammalian cancer cells (29); therefore, mechanisms associated with active efflux have become of considerable scientific interest. The first suggestion that the ribosome was the site of action of streptomycin came in 1961 (97). Since that time the ribosome has been positively identified as the target site for all the aminoglycoside-aminocyclitol drugs that have been tested. The binding of streptomycin to the ribosome is reversible (6, 16); however, the uptake of streptomycin is irreversible (15, 78). The binding of streptomycin to ribosome induces misreading and inhibition of protein synthesis. It has been proposed (15, 25) that the irreversible uptake and misreading (mistranslating) and inhibition of protein synthesis is associated with the bactericidal, not bacteristatic, action of streptomycin; however, the detailed mechanism of action is still unclear. Bacteria use the same strategies to cope with streptomycin as with other antibiotics and toxic elements. The mechanisms of resistance used for streptomycin include alteration of the ribosomal target site, inactivation by modifying enzymes, and reduced accumulation of streptomycin in the cells (5, 23). Biochemical and genetics studies have revealed that streptomycin primarily interacts with 168 ribosomal RNA (rRNA). Cross-linking experiments have shown that streptomycin can be linked to a fragment of Escherichia coli 168 rRNA spanning residues 892 to 917 and 1394-1415 (39), and chemical footprinting studies indicated that bases 909, 911, 912, and particularly bases 913 to 915, which have been designated as the 915 region, are protected by streptomycin (72). Streptomycin resistance can result from changes at the bases equivalent to E. coli 523 (designated 530 loop) and 912 to 915 in the 16S rRNA or changes of amino acid in ribosomal protein (r-protein) 84, 85 and 812. It has been observed that mutations at position 912, 914, or 915 (E. coli numbering) in chloroplast 16S rRNA confer resistance to streptomycin (42, 74), and mutations at position 912, 913, and 915 have been shown to lower the response of ribosomes to streptomycin when introduced into E. coli 16S rRNA (30, 73). Although the 530 loop and r- proteins 84, 85, and 812 are located on a distinct site from the streptomycin binding site (59, 69), they are also involved in streptomycin binding. A mutation at position 523 in the 530 loop of 16S rRNA has been proved to cause resistance to streptomycin (33, 69). Protein 812, when altered, can produce a phenotype of streptomycin resistance (37, 62, 81, 101) in bacteria and chloroplasts, or streptomycin dependence (9). Mutations in protein 812 confer streptomycin resistance by impairing the binding of the drug to the ribosome, whereas, mutations in protein S4 or 85 (ram mutations) have the opposite effect since they enhance streptomycin binding (10, 59). Aminoglycoside-modifying enzymes include acetyltransferases (AAC), nucleotidyltransferases (ANT) (adenylyltransferases, AAD), and phosphotransferases (APR) (23, 93). Among the enzymes two nucleotidyltransferases, ANT(3") and ANT(6), and two phosphotransferases, APH(3") and APH(6), are associated with resistance to streptomycin (93). ANT(3") confer by resistance to streptomycin and spectinomycin (24, 46). The enzyme modifies the 3"-hydroxyl position of streptomycin and the 9-hydroxyl position of spectinomycin. The ant(3") gene has been cloned in association with several transposons (46, 87) and is ubiquitous among gram-negative bacteria. ANT(6), APH(3") and APH(6) are characterized by resistance to streptomycin only. ANT(6) is found in gram-positive organisms (82) and reacts at the 6-hydroxyl position of streptomycin. APH(3") and APH(6) modify streptomycin at the 3"- and 6-hydroxyl groups, respectively. Both phosphotransferases have been found in streptomycin producing strains of Streptomyces griseus (45, 65), but APH(6) is the major enzyme and its gene is clustered with the genes encoding enzymes involved in streptomycin biosynthesis (65). APH(3") has been found in many clinically important gram—negative bacteria (38, 50, 80) and in phytopathogenic bacteria (36, 108). Because bacteria with reduced drug accumulation exhibit low-level resistance, whereas bacteria with altered ribosome and inactivating enzymes exhibit high-level resistance, the mechanism of reduced accumulation has not been studied in detail even though it is potentially clinical importance. Whether or not this mechanism results from the development of an active efflux system remains to be investigated. Horizontal and vertical transfer of resistance to drugs and toxic metal ions in bacteria has been shown to be mostly conveyed by plasmids and transposons which carry resistance genes (34, 51, 64, 92). Different species of bacteria harbor characteristic types of plasmids, some of which can mediate their own transfer by conjugation. However, except some groups of broad-host-range plasmids most plasmids can only exist and replicate themselves in a limited number of bacterial species (105). Transposons have the capacity to transpose from one DNA molecule to another. This has undoubtedly contributed to the rapid dissemination of antibiotic resistance by providing an efficient mechanism for incorporating resistance determinants into new plasmid vectors which can transfer to and stably replicate in diverse hosts. Recent studies on the horizontal transfer of tetracycline resistance in the gram-positive clinical pathogen, Streptococcus, have shown that several nonplasmid conjugative elements, designated conjugative transposons, mediate the transfer of resistance genes (17, 35, 90). Conjugative transposons transpose from donor (usually chromosome-borne) to recipient by a circular DNA intermediate rather than by a conjugative plasmid. Conjugative transposons may be more commonly involved in the 10 dissemination of drug resistance than plasmids in some species of the clinically important streptococci. Transposons have been divided into two groups based on the organization of their genes (51). Class I transposons, for example Tn5, carry genes for antibiotic resistance bounded by two directed or inverted copies of an insertion sequence (18) element in the form of a compound transposon. Class II transposons contain short inverted terminal repeats, a tnpA encoding transposase of about 1,000 amino acids, a tnpR encoding resolvase of about 185 amino acids, and drug or heavy metal ion resistance gene(s). Some class II transposons, especially those belonging to the Tn21 subfamily, also contain a gene encoding integrase which mediates a site-specific integration mechanism responsible for the acquisition of other resistance genes. Therefore many class II transposons carry several genes for multidrug resistance which has resulted in the failure of many antibiotics in chemotherapy (67, 100). The presence of a transposon is usually detected by the movement of the marker gene(s) to a recipient replicon, a small multi-copy plasmid or a conjugative plasmid with an selection marker (27, 35, 44, 99). The movement of transposon (transposition) is accomplished by transposase and site-specific recombinase (resolvase) that are encoded by the transposon (40, 92). Usually, a direct repeat in the target DNA is generated following the transposition. 11 It has been suggested that the aminoglycoside resistance genes in clinically important bacterial pathogens were derived from organisms that produce the aminoglycosides (8). The presence of these enzymes in aminoglycoside-producing strains could provide a mechanism of self-protection against the antibiotic produced. Therefore, the actinomycetes could have provided the initial gene pool from which some of the present-day aminoglycoside resistance genes were derived. A second theory is that aminoglycoside resistance genes are derived from bacterial genes which encode enzymes involved in normal cellular metabolism (83). Recent studies reveal that overexpression of a cellular gene may result in multiple antibiotic resistance in some bacteria (18, 94) as well as multiple resistance to anti-cancer drugs in mammalian cancer cells (30). The gene aac(6')-Ic was detected in both susceptible and resistant strains of Serratia, but the AAC(6') enzyme was overexpressed in resistant strains (94). Another example is the mar (multiple antibiotic resistance) locus in E. coli. Mar mutants overexpress Mar proteins that mediate the cross- resistance to several unrelated antibiotics and their genes have been found in other members of the family Enterobacteriaceae, including Salmonella, Shigella, and Klebsiella spp. (19). Although these genes are limited to a few genera, it is possible that further genetic selection will result in the mobilization of these genes via association with plasmids or transposons and will speed the 12 dissemination of the resistant determinants through species barriers. Although streptomycin resistance in phytopathogenic bacteria have been noticed since 1958 (31), the mechanism of resistance was not studied until recent years. Schroth et al. assumed that resistance to streptomycin in the strains of E. amylovora was caused by a chromosomal mutation because no plasmid could be detected (89). Later, resistance in P. syringae pv. papulans and X. campestris pv. vesicatoria were found to be carried on plasmids (13, 71). A specific probe, 8MP3, cloned from P. syringae pv. papulans strain Psp36 was developed for detecting the occurrence, frequency, and distribution of the resistance gene in the pathogen and many epiphytic bacteria in New York apple orchards (79). The probe also hybridized to plasmids in distinct sizes in the epiphytic bacteria suggesting that different plasmids had carried the same resistant determinant (79). Probe 8MP3 was also used for a study on streptomycin-resistant E. amylovora and epiphytic bacteria isolated from Michigan apple orchards (96). This study indicated that 97% of 152 strains of epiphytic gram-negative bacteria contained DNA homologous to the DNA associated with resistance in P. syringae pv. papulans but none of 28 gram-positive bacteria contained DNA that hybridized with SMP3. Restriction fragment length polymorphism of the resistance gene was present among the resistant epiphytic bacteria and one resistant strain of E. amylovora (79, 96). The resistance determinant in the 13 streptomycin- and copper-resistant P. syringae pv. syringae has also been studied (102). A restriction mapping indicated that organization of resistance genes in P. syringae pv. syringae strain A2 was homologous to streptomycin resistance genes strA-strB in plasmid R8F1010 (88, 102). The appearance of antibiotic-resistant human pathogenic microbes has become a worldwide crisis in this decade. Rapid development of resistance, especially multidrug resistance, has stun the pharmaceutical industry in creating new antibiotics and caused the failure of many antibiotics in treatment (61, 76). The deciphering of drug resistance mechanisms will not only allow a better understanding of incipient clinical crises but may also suggest strategies for reversing resistance and preventing the appearance of new resistant microbes (107). Similarly, the reasons for studying streptomycin resistance mechanisms in E. amylovora are to develop a better understanding of the problem and methods for combating resistant strains. In Michigan, streptomycin-resistant strains of E. amylovora were first isolated from an apple orchard in southwest in the summer of 1990. More resistant strains of E. amylovora and many epiphytic bacteria were collected from the orchards in the same area and Grand Rapids area in 1990 and 1991. The objective of this research is to determined the mechanism of resistance in the streptomycin-resistant E. amylovora strain CA11 on a molecular genetic and biochemical 14 basis. Part I describes the molecular genetic analysis of the resistance determinant. 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A survey of Xanthomonas vesicatoria resistance to streptomycin. Proc. Fla. State Hortic. Soc. 75:163-165. 105. Thomas, C. M. (ed.). 1989. Promiscuous plasmids of gram-negative bacteria. London, San Diego, Academic Press, 276 pp. 106. van der Zwet, T., and n. L. Eeil. 1979. Fire blight: A bacterial disease of Rosaceous Plants. U. 8. Dep. Agric. Agric. Handb. 510. 200pp. 107. Walsh, C. T. 1993. Vancomycin resistance: decoding the molecular logic. Science 261:308-309. 108. Yano, E., H. Fujii, H. Mukoo, M. Shimura, T. Watanabe, and Y. Sekizawa. 1978. On the enzymatic inactivation of dihydrostreptomycin by Pseudomonas lachrymans, cucumber angular leaf spot bacterium: isolation and structural resolution of the inactivated product. Ann. Phytopath. Soc. (Japan) 44:413-419. 109. Young, J. M. 1977. Resistance to streptomycin Pseudomonas syringae from apricot. N. Z. J. Agric. Res. 20:249-251. PART I THE ANALYSIS OF PLASMID-MEDIATED STREPTOMYCIN RESISTANCE IN Erwinia amyl ovora Disease Control and Pest Management The Analysis of Plasmid-Mediated Streptomycin Resistance in Erwiuia amylovora Chien-Shun Chiou and A. L. Jones Department of Botany and Plant Pathology and the Pesticide Research Center, Michigan State University, East Lansing 48824- l3l2. This research was supported in part by the Michigan Agricultural Experiment Station. We thank J. L. Norelli for providing the cloned streptomycin-resistance gene, T. J. Burr for strains of Pseudomonas syringae pv. populam, R. G. Roberts and J. L. Norelli for strains of Erwinia amylovora, G. R. Ehret for technical assistance during the early stages of this study, and P. Sobiczewski for useful discussions during the course of this study and for assistance in preparing colony blots for probing. Accepted for publication 19 February 1991 (submitted for electronic processing). ABSTRACT Chiou, C.—S., and Jones, A. L. 1991. The analysis of plasmid-mediated streptomycin resistance in Erwint'a amylovora. PhytOpathology 81:710-714. Streptomycin-resistant mutants of Erwim‘a amylovora were isolated from an apple orchard in Michigan and from crabapple trees adjacent to the same orchard in 1990. Isolates that grew on King's medium 8 amended with 100 pg/ ml of streptomycin sulfate were considered to be resistant strains, whereas isolates that failed to grow on this medium were considered to be sensitive strains. Growth of the resistant strains was not inhibited in a filter-paper disk assay (0.06-5 pg of streptomycin sulfate). but growth of sensitive strains was inhibited at concentrations as low as 0.06 pg of streptomycin sulfate. Only sensitive strains were detected in an additional 19 apple orchards sampled for resistant strains. In colony blot hybridizations, an internal portion of the streptomycin- resistance gene (probe SMP3) from strain Psp36 of Pseudomonas syringae pv. papulans hybridized with all streptomycin-resistant strains of E. amylovora, but not with streptomycin-sensitive strains. Probe SMP3 hybridized to a 2.7-kb restriction fragment from Avril-digested total genomic and plasmid DNA of two resistant strains of E. amylovora and to a LS-kb fragment in DNA from strain Psp36 of P. s. papulans. The probe did not hybridize with digested DNA from sensitive strains. A 33-kb plasmid was present in all streptomycin-resistant field strains but not in streptomycin-sensitive strains. Streptomycin resistance was transferred by matings to four streptomycin-sensitive recipient strains of E amylovora from each of two streptomycin-resistant donor strains. Transconjugants also contained the 33-kb plasmid. DNA from resistant strain Ea88-90 from Washington did not hybridize with the probe, indicating that this strain contains a resistance system unrelated to that in streptomycin-resistant strains from Michigan. Fire blight. caused by Erwim‘a amylovora, is a devastating disease of apples and pears in North America, New Zealand, much of Europe, and the Mediterranean region (I). Extensive experimentation on the efficacy of streptomycin for the control of fire blight was conducted in North America in the early 19505. After streptomycin was registered in the United States in the late [9505, it was used extensively for the control of fire blight on pears in the West and on apples in the East. Streptomycin-resistant E. am ylovora was first detected in pear orchards of California in l97l (l4) and soon thereafter in pear orchards of Washington and Oregon (4); in 1988, resistant strains were ubiquitous in pear orchards of Washington (I I). In the mid- l970s, attempts to detect resistant strains in apple and pear orchards in western New York state and in apple orchards in Michigan failed (2,22). Except for a report of resistant strains on apple in Missouri (19), detection of streptomycin-resistant E. amylovora has been limited to the western United States. How- ever, streptomycin resistance in Pseudomonas syringae pv. papulans has been a problem in apple orchards of the cultivar Mutsu in the eastern United States (3,8,17). In California, resistance to streptomycin in E. amylovora is caused by a chromosomal mutation (18), but streptomycin resis- tance in P. s. papulans and Xanthomonas campestris pv. vesicatoria is plasmid-borne (3.15.17). No hybridization was obtained when a streptomycin-resistance probe from X. c. vesicatoria was used to probe DNA from a streptomycin-resistant strain of E. amylovora from California (l5). But the resistance probe did hybridize with DNA from a streptomycin-resistant strain of P. s. papulans from New York. This study reports the detection of streptomycin-resistant E. amylovora on apple in Michigan. It shows that resistance is 0 1991 The American Phytopathological Society 710 PHYTOPATHOLOGY plasmid-borne, and that a DNA probe specific for streptomycin- resistance in P. s. papulans hybridizes with DNA from strepto- mycin-resistant strains of E. amylovora but not with DNA from streptomycin-sensitive strains. MATERIALS AND METHODS Bacterial strains and plasmids. Streptomycin-resistant strain Ea88—90 of E. amylovora was supplied by R. G. Roberts, Tree Fruit Research Laboratory, Wenatchee, WA. All other strains of the bacterium were isolated from apple trees in Michigan during the course of this study. Strains Psp36 and Psp32 of P. s. papulans were supplied by T. J. Burr, New York State Agricultural Experi-. ment Station, Geneva. Plasmid pCPPSOS, containing an internal portion of the streptomycin resistance gene from strain Psp36 of P. s. papulans (17), was supplied by J. L. Norelli, New York State Agricultural Experiment Station, Geneva. Detection of streptomycin-resistant Erwinia amylovora. In June 1990, samples of fire blight were received from an apple grower in southwest Michigan (orchard SW) who suspected a problem with streptomycin-resistant E. amylovora. Bacteria were isolated from the samples by placing small bits of tissue from infected spurs and shoots on King‘s medium B (KB) containing 100 pg/ml of streptomycin and 50 pg/ml of cycloheximide (KBsc). The pathogenicity of eight strains isolated from orchard SW was established by inoculating immature Jonathan apple fruit in the laboratory. Upon reisolation, five strains from orchard SW and two strains from an orchard located at the Botany and Plant Pathology Farm, Michigan State University, East Lansing, were tested for resistance to streptomycin by evenly spreading 107 colony-forming units per milliliter onto nutrient-yeast-dextrose agar (4). About 5 h later, six 12.7-mm-diameter filter-paper disks (Schleicher & Schuell Inc., Keene. NH) were placed equidistant on the surface of the plates. Aliquots (50 pl) of solution from a streptomycin sulfate dilution series were applied to the disks. 26 27 Each treatment was replicated four times. Plates were incubated at 22 C, and inhibition zones were measured after 4 days. Orchard survey. Spurs and terminals with active fire blight lesions were collected in June and early July 1990 from 20 apple orchards throughout the western Michigan fruit belt, including orchard SW. Infected twigs were also collected from seedling crabapple trees in a 2-ha block adjacent to orchard SW. After pulling back the bark, two small pieces of tissue were removed from the water-soaked tissue distal to the necrosis. One piece of tissue was placed on KB with 50 pg/ ml of cycloheximide (KBc), and the second was placed on KBsc. Isolations were made from up to 10 samples per location (25 samples, however. were taken from orchard SW). Nonfluorescent white, semimucoid colonies typical of E. amylovora that formed on KBc or KBsc in 72 h at 20 C were streaked onto the selective high-sucrose medium of Crosse and Goodman (5). Bacteria characteristic of E. amyla- vora on the high-sucrose medium were saved for study. Plasmid characterizations. Sensitive and resistant strains of E. amylovora from orchard SW and from crabapple trees adjacent to orchard SW were screened for plasmids using a modification of the method of Kado and Liu (9) as described by Burr et al (3). Plasmid DNA was electrophoresed on 0.5% agarose gel, stained, and photographed as described by Sundin et a1 (21). Hybridizations. Plasmid pCPP505 was received, maintained, and amplified in Escherichia coli strain DI-Il. Probe SMP3 was prepared as described by Norelli et a1 (17). Plasmid DNA, extracted by the alkaline lysis method described by Maniatis et a1 (12) from cultures grown for 16 h at 37 C in Luria-Bertani (LB) medium containing 50 pg/ml of ampicillin and 50 pg/ml of streptomycin, was digested with restriction enzymes BamHI and Aval. A 500-bp restriction fragment (SMP3) was excised from a low melting temperature agarose gel following electrophoresis in Tris-borate EDTA buffer. The fragment was radiolabeled with 32P by the randomized oligonucleotide labeling procedure (Random Primed DNA Labeling Kit, U.S. Biochemical, Cleveland, OH). Colony hybridizations with DNA probe SMP3 were performed with streptomycin-sensitive and streptomycin-resistant E. amylovora. Up to five sensitive strains from each orchard and all resistant strains were transferred to Colony/ Plaque Screen hybridization transfer membranes (New England Nuclear Research Products, Boston, MA) that had been placed on the surface of KB agar plates and incubated for 48 h at 20 C. Colonies of E. coli with pCPP505 and strain Psp36 of P. s. papulans (strep- tomycin-resistant) and strain Psp32 of P. s. papulans (strepto- mycin-sensitive) were included on each membrane as positive and negative controls, respectively. The bacteria were lysed and the DNA denatured and fixed to the membranes according to the manufacturer’s instructions. Hybridizations were performed over— night and the membranes washed according to the manufacturer‘s recommended procedures. Autoradiographs of membranes were carried out with XAR X-ray film at -70 C. Restriction enzyme digests and Southern blots. Plasmid DNA was isolated by alkaline lysis extraction followed by cesium chloride centrifugation (12). Total genomic DNA was prepared by a miniprep procedure (23). The purified plasmid and total genomic DNAs were digested with AMI, and following gel electro- phoresis and Southern transfer to GeneScreen hybridization transfer membrane, hybridizations were carried out as described for colony hybridizations. Bacterial conjugation. Recipient strains were rifampicin- resistant variants of streptomycin-sensitive parental strains ELOl, BC06, GR05, and MAOS. Donor (11062-1 and CA11) and recipient strains were grown for 16—24 h at about 22 C on a rotary shaker in 5 ml of LB medium amended with 50 pg/ ml of streptomycin (donor strains) or 150 pg/ml of rifampicin (recipient strains). The cultures, each with about 10” cells per milliliter, were mixed in a 1:1 ratio, and 10-p1 aliquots were plated on KB medium and incubated 24 h at 22 C. Cell mixtures were suspended in 10 ml sterile distilled water, vortexed, serially diluted, and plated on LB medium amended with 50 pg/ m1 of streptomycin or 150 pg/ ml of rifampicin to determine donor and recipient populations, respectively. Cell mixtures were also plated on LB medium amended with both 50 pg/ml of streptomycin and 150 pg/ ml of rifampicin to determine the population of trans- conjugants. Colonies with good growth on LB medium amended with both antibiotics were considered putative transconjugants. The frequency of spontaneous resistant mutants was determined by plating donor and recipient strains on LB medium amended with rifampicin and streptomycin, respectively. RESULTS Detection of resistance. Colonies of E. amylovora were recovered on KBsc medium from eight samples of fire blight from orchard SW. When immature fruit were inoculated with these bacteria, all of the strains caused typical symptoms of fire blight, including the production of bacterial ooze. Level of resistance. Pathogenic strains initially recovered on KBsc medium were highly resistant to streptomycin. Each strain grew to themargin of filter-paper disks containing the highest level (5 pg per disk) of streptomycin sulfate tested. Two strains from the orchard in East Lansing were sensitive to streptomycin, as indicated by the development of clear zones around each disk. Mean size of zones of inhibition for the two sensitive strains were 13.6, 14.2, 15.8, 19.4, 22.2, and 25.0 mm for disks that received 0.06, 0.31, 0.62, 1.25, 2.50, and 5.0 pg of streptomycin sulfate, respectively. Distribution of resistant strains. E. amylovora was recovered on KBc from 137 samples and on KBsc from 20 samples of fire blight collected from 20 apple orchards in western Michigan. All streptomycin-resistant strains (20 out of 25 samples) were recovered from orchard SW. No streptomycin-resistant strains were detected in samples from the remaining 19 orchards. Five streptomycin-resistant strains of the bacterium were also recovered from 15 samples of fire blight collected from crabapple trees adjacent to orchard SW. Colony hybridization studies. The probe hybridized with plasmid pCPP505 and with DNA from streptomycin-resistant strain Psp36, but not with DNA from streptomycin-sensitive strain Psp32 of P. s. papulans. Total DNA from all 25 streptomycin- resistant colonies of E amylovora from orchard SW and the adjacent crabapple trees (but no DNA from 78 colonies of strepto- mycin-sensitive E. amylovora, including five strains from orchard SW) hybridized with the probe in colony blot hybridizations. Probe SMP3 did not hybridize with DNA from streptomycin- resistant E. amylovora strain Ea88-90 isolated from pear in Washington. Plasmid characterizations. Streptomycin-resistant strains from orchard SW and the neighboring crabapple trees contained a plasmid of approximately 33 kb that was not present in strepto-' mycin-sensitive strains isolated from these same locations (Fig. 1). In addition, all strains from these orchards and all other strains of E. amylovora examined for plasmid content, including strain Ea88—90 from pear in Washington state, contained a plasmid of approximately 30 kb. Confirmation that this plasmid was the ubiquitous plasmid common to E amylovora (10) was obtained by comparing fragment sizes from Sail, Psrl, and Kpnl restriction digests of the isolated plasmid with fragment sizes reported in Table l of Falkenstein et a1 (7). Single digests with each of these restriction enzymes of the plasmid isolated from strain Ea88—90 yielded 4, 9, and 5 restriction fragments, respectively. Strepto- mycin-sensitive strain BC06 contained a large plasmid in addition to the 30-kb plasmid. Southern analysis. A single 2.7-kb fragment in Aval digests of both plasmid and total genomic DNAs of two streptomycin- resistant strains of E. amylovora hybridized with probe SMP3 (Fig. 2, lanes 5, 6, 9, and 10). None of the Avril fragments of DNAs from two streptomycin-sensitive strains of E. amylovora hybridized with the probe (Fig. 2, lanes 7, 8, 11, and 12). Hybrid- ization with probe SMP3 also occurred with a 1.5-kb fragment in Aval digests of plasmid and of total genomic DNAs from streptomycin-resistant P. s. populous strain Psp36 (Fig. 2, lanes 1 and 2) and with a 3.7-kb fragment in digested DNA: from Vol.81, No.7, 1991 711 E coli with plasmid pCPP505 (Fig. 2, lanes 3 and 4). Bacterial conjugation. Transfer of the plasmid carrying the gene ntaneous mutations to streptomycin resistance. were less than 2.0 X 10' , and spontaneous mutations to rifampicin resistance was about 5 0 X 10" per donor cell. Transfer of streptomycin resistance in E. 1ylovora was associated with transfer of the 33- kb plasmid Au transconjugants resulting from the mating of donor strains CA” and recipient nsrBC06 and ELOI rif‘ contained a 33- kb plasrni id not present in the recipient strains (Fig.3). DNA of putative trans- conjugants, randomly chosen from ant tibi otic selection pla a,tes always co nt tained an Aval restrict ion fragment identical to one in restriction digests of DNA Aofr om donor strains but absent in restriction Ndigests of DNA Arfom recipient strains (Fig 4.) las midD Afromtransconjugant BC06 rif’X HO62- 1 contained a 23- kb restriction fragm associated with donor H062- l and CA11 but notw with recipient BC06 rif' (Fig. 4A lanes 4-6). A 2. 7- kb Aval restriction fragment in streptomycin- resistant donor and transconjugant strains but not in streptomycin- sensitive recipient strains, hybridized with probe SMP3 (Fig. 4B, lanes 2—4, 6, and 7) kb 33 30 n‘IIPh‘E'h-rla - ru .1 . - . ‘frnmnpple orchard SW in southwest Michigan androf madjacent crabapple trees. Streptomycin-resistant strains CAI3, CAII, HT16, HTOI- l. and HT06 are in lanes 1, 3, 6, 7. and 8,1upectively, and streptomycin-sensitive strains CAIO, CA06. HTOJ, and HTO nlanes 2, ,,9 and 10, respectively. Lane 5 contains lenia slewam‘i strain S 7..4 123456789101112 ltb 3.7— .- 2_7 -- “ 1.5——- Flg. 1. Autoradiograph of a Southern blot of plasmid DNA. first, and total genomic" DNA (including plasmid DNA). second, of Prendomanm syringae pv.); pularu strain Psp36 (lanes 1 and 2).:1nd DHl with plasmid pCPP505 (lanes 3 andn 4) and streptomtfin resista ntC( All. lanes 5 and 6. H 0,62-1 lanes 93 le) ands mycm- susceptible (BC06. la Inc: 7 and 8, ELOI, lanes nll and l2) itraipns of Erw1n1'a army/01am digested with AvaI and hybridized to probe” SMP3 The sizes (in lib) of hybndizing _, laui‘uua DNA are given at the far left. 712 PHYTOPATHOLOGY 28 DISCUSSION The detection of a wputative streptomycin- -resistance gene in E amylovora that gre on media mended with streptomycin is strong evidence that streptomycin- resistant strains of the bacterium are present in Michigan .In addition, our conjugation and hybridization studies provide evidence that tiress tance in E. :mylovora from Michigan is plasmid- -bome. In our study, probe SMP3 did not hybridize with DNA from streptomycin- -resistant strain Ea88- 90 of E. amylotora from Washin ngto .‘IWie fi of probe SMP3 to hybridize with a streptomycin- -resistant strain from the western United States is evidence that the resistance system in the two regions is unrelated. NA . 1 1 Michigan hybridized with DNA sequences from the internal portion of a streptomycin- resistant gene cloned from strain Psp36 of P. J. papu lam. In pre ous studies on streptomycin resistance in field strains of E. amylovom from California, resistance was ntd obe chromosomal (18). In addition, a 4.9- kb DNA subclone from streptomycin- resistant X. c. vesicatoria did not hybridize with DNA from streptomycin— resistant strain UCBPP 829 of E 11111on ovora from California, but it did hybridize with DNA from strain Psp36 of P. s. papulans from New York (15). This is evidence that the DNA sequences in the two pathogens are closely related. Base sequencing and other forms of genetic analyses are needed to establish whether the genesi teh pathogens are identical Because the DNA sequences associated with resistance in P. s. papulanr and X. c. vesicatoria are related (IS), the DNA associated with resistance in E. amylovora must also be closely related to the DNA associated with streptomycin- resistance in X c. vesicatoria.- Tk . p ' ‘ 1. A SW and in crabapple trees next to orchard SW. but not in 19 other orchards, is evidence that resistant strains of the bacterium are currently localized in Michigan‘s large apple-growing region. The high frequency of transfer of streptomycin resistance from donor strains H062-l and CA” is evidence that transfer of resistance to other strains of the bacterium could result in a rapid increase in streptomycin-resistant E amylovora in Michigan orchards. The spread and buildup of streptomycin-resistant E. Heathen...” ' ;.,' f4 ' or ' ' . J ‘ r J Donor strains Recipient Recipient strains HO6‘- CAI I means £1.01 .3x 10" 27x 10 2.8x lo" BC06 61X 10" 33x10“ 50x10“ GRos 32x 10'1 10x10" 21 x 10" MA05 45x10" 25x10“ 3 5 ><1o*1 .3 Agarose gel electrophoresis of cleared lysates ofdonor.recipien1 and transconjugant strains of Erwmia am_1[otora.Lanel rec1pient BC06 rif: lane 2 transconjugant BC06 nf' X CA11; lane], donor CA11 lane lrif' X CAII; lane 5, rec1pient ELOI rnf'; lane 6. strain Ea88-90WI'I'10'“71 29 “ ~ .r- .1. Flg. 4. Ethidium bromide-stained gel (A) and autoradiograph of a Southern blot (B) of plasmid DNAs of donor. recipient and transchnjugant struns tramsof Erwinia amylavora digested with Aval and hybridized to pro obe SMP3. X CAII; lane 3. donor iCAll; lane 4, otransconjugant BC06 rif'X Ian: 7 donor e.8 tran rif' X HO62- I; CAII: lane 5. recipient BC06 rif'; lane 6. transconjugant tBC06 C06rif'X 0Ll lrif' H062- l; e.9 recipient ELOI rif'. The sizes (in Itb) of hybridizing fragments Lane I, recipient ELOI rif'; lane 2, transconjuganth and In dated from EeoRlI- Hind]II-digesstcedn bacteria Ephnlge lambda DNA are given at the far left amylovora presents a serious economic threat to apple and pear grow rsbecause of teh lack of alternative bactericides for th control of fire blight. TolIm ‘ tfurthcr spread of resistant strains and reduce the likelihood of selecting resistant strains else where in Michigan it is important that apple and pear growers limit applications of streptomycin to a period from bloom to about the first cover stage ud development (a maximum of four applications per ae.ason) The frequency of spray applimcationis could be reduced even more by usin ga afor ssty nthe timing of applications of streptomycin for thee control of fire blight (20) Although the occurrence of streptomycin- resistant E. amylovom in California and Washington couldn at be correlated with the use of streptomycIn( l I,l8), It was suggested that selection pressure was not sufficient in the East, due to the limited use of strepto- rr.|.ycin to cause resistant strains to build up to detectable levels (l6). Orchard SW has had a history of fire blight problems for over 20 yr. and the management program or Ire blight in this orchard has been one of the most intensive in Michigan (A. L. Jones, personaI observation). Grower records indicate that in I988, I989,sn vely. The continued and frequent use of streptomycin in orchard SW could have resulted in the buildup of streptomycin- resistant E. amylovora to detectable levels Although Terra yCI stre reptomycin combinations ave ens own to delay resistance in laboratory trials (6), this strategy cannot be utilized until Terramycinw is registered on apples edetected streptomycin resistant E. umvlovora in crabapple trees. the ere is evidence that the resist ant st rains hav ve started! trno ove out of orchar d.SW Now these infected crabapple trees are a potential source of resistant strains for reinfecting orchard SW and for infecting other orchards currently free of resistant strains. Removal of these crabapple trees would reduce the potential for further movement of resistant strains in southwest Mic chigan It is also likely that the resistant strains in the two orchards have a common source. Strain H062- I from orchard SW and strain CAI I from crabapples carried the resistance gene on the same size plasmid. Also no restriction— —length poly— mo orphism was observed among Aval DNA fragments from the two strains. but Aval digests of DNA from some sensitive strains produced different restriction patterns. Unlike P. s. populous. with its high diversity in plasmid content (3.8). the plasmid content of van: is predictable and not diverse. All strains have a plasmid of approximately 30 kb. and some strains have an additional plasmid of approximately 65 kb (13). The failure to detect a plasmid of approximately 33 ltb in earlier studies on plasmids in E. amylavora suggests that it may be a recent introduction into the bacterium It 13 possible that a transposable element carrying the resistance gene has een inserted into the approximately 30—lt b pl .or that E amyla- vora has acquired a new p asrn d.Characterization of the plasmid of approximately 33 kb would dhelp to determine if one of these possibilities is correct. sThe streptomycin-resistance gene identified in E. amylovora widely geographically distributed in P. s. papulans and in X.c vesicatoria (3, 8. l5). It Is also widely distributed among a diverse group of gram-negative bacteria isolated from apple orchards an York (l7) and in Michigan. includin ng orchard SW (P. Sobiczewski, C. S. Chi Iou, andA. L Jones, unpublisheddala). The presence of a homologous streptomycin- resistance gene in both saprophytic and pathogenic bacteria that inhabit bapple suggests that acquisition of str tomycin resistance by E. aim I’IDVOI‘a was by conjugation. We are currently investigating the possibility that the gene can be transferred from saprophytic gram- negative bacteria to E am} lav oar LITERATURE CITED . Beer.S .l.990 Fire blight. Pages 61-63 In: Compendium of Apple and Peaerise cues AL. Jon andl H. S. A.ldenckle eds. American Phytopathological Society St.P Bcc ccr. S V.. and Norelli. J L I976. Streptomycin- -resistant Erwima aim/ovora not found In western New Yorlt pear and apple orchards N Plant Dis. Rep ()0l ‘6. 3, Burr. T J.. Nor .J L. Kat 1MB Wilcox F and Hoying. S. A 988 Strepltlomycin resistance of Pseudowmonas syrmgae pv. papa/ans in apple rcha rsd and its assocmtion wuh a conjugative pla asmid. Phytopathology 78; 4al0-4I3. . Coerr. D L. and Covey,R P I975 Tolerance of ErII-Iniaamylovom to streptomycm sulfate In Oregon and Washington Plant Di .Rep. 59:849-852 Cnrosse. J. E..a nd Goodman. R. N 73 A sclec ctIve medium for a definitive collony characteristic of Emim'a arm/ovora. Phyto pathologv 6314245 26. 6. En nglh.s gyA. R. and Van Halsema. G as 3" I954. A note on the delay Vol.81,NoI7.1991 713 I3. 14. 714 30 in emergence of resistant Xamhomonas and Erwinia strains by the use of streptomycin plus terramycin combinations. Plant Dis. Rep. 38:429-43l. . Falkenstein, IL, Bellemann, P., Walter, S., chlcr. W., and Geider, K. I988. Identification of Erwim'a amylovora, the fireblight pathogen, by colony hybridization with DNA from plasmid pEA29. Appl. Environ. Microbial. 54:2798-2802. . Jones, A. L., Norelli. J. L., and Ehret, G. R. 1991. Detection of streptomycin-resistant Pseudomonas syringae pv. populous in Michigan apple orchards. Plant Dis. 75:529-531. . Kado, C. I., and Liu, S. T. l98l. Rapid procedure for detection and isolation of large and small plasmids. J. Bacterial. “521365- I373. . Laurent, J., Barny, M.-A., Kotaujansky, A., Dufriche, P., and Vanneste, J. L. I989. Characterization of a ubiquitous plasmid in Erwim'a amylovora. Mal. Plant-Microbe Interact. 2:160-l64. . Loper, J. E., Henkels, M. D., Roberts, R. 6., Grave, G. 6., Willett, M. J., and Smith, T. J. l991. Evaluation of streptomycin. oxytetra- cycline, and copper resistance of Erwim'a amylovora isolated from pear orchards in Washington State. Plant Dis. 75:287-290. Maniatis, R., Fritsch, E. P., and Sambrook, J. I982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 545 pp. Merckaert, C., Thiry, M. E., Thiry, G. J., and Ledous, L. I982. Characterization of the plasmids present in Erwinia am ylovora. Arch. Int. Physiol. Biochem. 90:854-855. Miller, T. D., and Schroth, M. N. 1972. Monitoring the epiphytic population of Erwinia amylavora on pear with a selective medium. PHYTOPATHOLOGY I8. I9. 21. i 22. 23. Phytopathology 62:] I75-1182. . Minsavage, G. V., Canteros, B. 1., and Stall, R. E. I990. Plasmid- mediatcd resistance to streptomycin in Xanthomonas campestris pv. vesicatoria. Phytopathology 80:719-723. . Mailer, W. J., Schroth, M. N., and Thomson. S. V. I98l. The scenario of fire blight and streptomycin resistance. Plant Dis. 65:563-568. . Norelli, J. L., Burr, T. J., Lo Cicero, A. M, Gilbert, M. T., and Kata, B. H. l99l. Homologous streptomycin resistance gene present among diverse gram-negative bacteria in New York apple orchards. Appl. Environ. Microbiol. 57:486-49l. Schroth, M. N., Thomson, 8. V., and Moller, W. J. I979. Streptomycin resistance in Erwinia amylovora. Phytopathology 69:565-568. Shaffer, W. H., and Goodman, R. N. I985. Appearance of strepto- mycin-resistant Erwinia amylovora in Missouri apple orchards. (Abstr.) Phytopathology 75:]28I. . Steiner, P. W. I990. Predicting apple blossom infections by Erwinia am ylovora using the Maryblyt model. Acta Hortic. 273:139-148. Sundin, G. W., Jones, A. L., and Fulbright, D. W. 1989. Copper resistance in Pseudomonas syringae pv. syringae from cherry orchards and its associated transfer in vitro and in planta with a plasmid. Phytopathology 79:861-865. Sutton, T. B., and Jones, A. L. I975. Monitoring D'wlnia amylovom populations an apple in relation to disease incidence. Phytopathology 65: 1009-1012. Wilson, K. I988. Preparation of genomic DNA from bacteria. Pages 2.4.I-2.4.5 in: Current Protocols in Molecular Biology. Vol. I. F. M. Ausubcl, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl, eds. John Wiley & Sons, New York. PART II NUCLEOTIDE SEQUENCE ANALYSIS OF A TRANSPOSON (Tn5393) CARRYING STREPTOMYCIN RESISTANCE GENES IN Erwinia amylovora AND OTHER GRAN-NEGATIVE BACTERIA JOURNAL or BACTERIOLOGY, Feb. 1993, p. 732—740 0021-9193/93/030732-0950200/0 Copyright O 1993, American Society for Microbiology Vol. 175, No. 3 Nucleotide Sequence Analysis of a Transposon (Tn5393) Carrying Streptomycin Resistance Genes in Erwinia amylovora and Other Gram-Negative Bacteria C.—S. CHIOU AND A. L. JONES‘ Department of Botany and Plant Pathology and the Pesticide Research Center, Michigan State University, East Lansing, Michigan 48824-1312 Received 30 July 1992./Accepted 23 November 1992 A class II Tn3-type transposable element, designated Tn5393 and located on plasmid pEa34 from streptomycin-resistant strain CA11 of Envinia amylovom, was identified by its ability to move from pEa34 to dill'erent sites in plasmids pGEM3Zf(+) and pUCDBOO. Nucleotide sequence analysis reveals that Tn5393 consists of 6,705 hp with 81-bp terminal inverted repeats and generates S-bp duplications of the target DNA following insertion. Tn5393 contains open reading frames that encode a putative transposase (my!) and resolvase (tnpR) of 961 and 181 amino acids, respectively. The two open reading frames are separated by a putative recombination site (res) consisting of 194 bp. Two streptomycin resistance genes, strA and arB, were identified on the basis of their DNA sequence homology to streptomycin resistance genes in plasmid RSF1010. StrA is separated from tnpR by a 1.2-kb insertion element designated 181133. The tnpA-res-tnpR region of Tn5393 was detected in Pseudomonas syringae pv. papulans Psp36 and in many other gram-negative bacteria harboring strA and strB. Except for some strains of Erwinia herbicola, these other gram-negative bacteria lacked insertion sequence 181133. The prevalence of str-A and mi? could be accounted for by transposition of Tn5393 to conjugative plasmids that are then disseminated widely among gram-negative bacteria. The bacterium Erwinia amylovora (Burrill 1882) Winslow, Broadhurst, Buchanan, Krumwiede, Rogers, and Smith 1920 causes fire blight, a disease of apple and pear trees and other rosaceous plants. Streptomycin has been used commercially in the United States, Canada, and New Zealand since the late 19505 to control fire blight and certain other bacterial diseases of plants. Streptomycin-resistant E. amylovora has been detected in fruit-growing areas of the western and midwestem United States (3, 4, 33, 34), but little is known about the molecular basis for the resistance in this bacte- rium. The development of resistant strains of E. amylovora is particularly significant because of the lack of alternative methods of control for this devastating disease. Resistance to streptomycin in Pseudomonas syringae pv. papulans, Xanthomonas compestn’s pv. vesicatoria, and E. amylovom is often plasmid borne (2, 3, 14, 24). Sequences of plasmid DNAs from these streptomycin-resistant bacteria and from numerous epiphytic gram-negative bacteria iso- lated from apple orchards cross-hybridized (3, 24, 27, 36). Although the DNA sequences associated with streptomycin resistance in these bacteria were related, the size of the plasmids harboring these sequences varied markedly. The widespread distribution among plant-pathogenic and plant-associated gram-negative bacteria of related plasmid- borne genes for resistance raises the basic question of how a common determinant for streptomycin resistance has devel- oped on different plasmids. Spread of antibiotic resistance by transposable elements in the bacterial population of human and animal pathogens is well documented (25), but to date no active transposable element has been demonstrated in the movement of antibiotic resistance among plant-patho- genic bacteria. We report that the genes for streptomycin resistance in E. ' Corresponding author. 732 32 amylovora are located on a transposable clement designated Tn5393 and that resistance in P. syringae pv: papulans Psp36 and in many epiphytic gram-negative bacteria from apple orchards is associated with Tn5393. In addition, we report that the nucleotide sequence of the genes conferring resis- tance to streptomycin in E. amylovora and epiphytic gram- negative bacteria from apple orchards is homologous to the streptomycin resistance genes on plasmid RSF 1010 (10, 32). MATERIALS AND METHODS Bacterial strains and plasmids. The bacteria and plasmids used in this study are listed in Table 1. Strains of Escherichia coli were grown on Luria-Bertani (LB) agar or in LB broth at 37°C. All other bacteria were grown on King’s medium B (16) agar or in broth at 22°C. Unless otherwise indicated, the media were supplemented with appropriate antibiotics at concentrations in micrograms per milliliter as follows: strep- tomycin, 50; kanamycin, 50; and ampicillin, 100. Hybridization studies. Colony and Southern hybridizations were performed by using Colony/Plaque Screen and Gene- ScreenPIus (New England Nuclear, Boston, Mass.) as de- scribed by the manufacturer. Probes were radiolabeled with [a-32P]dCTP by the randomized oligonucleotide labeling procedure (Random Primed DNA Labeling Kit, United States Biochemical, Cleveland, Ohio). Hybridizations were performed overnight, and the membrane was'washed ac- cording to the manufacturer’s recommended procedures. Autoradiography was performed at -70°C with XAR X-ray film. Cloning, isolation, and restriction mapping of pEa34. Plas- mid pEa34, which carries the genes for streptomycin resis- tance, was introduced into E. coli JMIO9 by conjugation from E. amylovora CA11 as previously described (3). Trans- conjugants were selected on LB agar supplemented with streptomycin and incubated at 37°C. The presence of plas- 33 VOL. 175, 1993 NUCLEOTIDE SEQUENCE ANALYSIS OF Tn5393 733 TABLE 1. Bacterial strains and plasmids used in this study Strain(s) or plasmid Relevant characteristiqs)‘ Source or reference(s) Bacterial strains Erwinia amylovora E1501, 8016 Sm‘ 3 CA11 Sm', containing pEa34 3 Escherichia coli .1le end/11 recAl gyrA96 thi hstl7(rK' mK‘) rel/11 supE44 A" AUac-pmAB) 44 [F ' tmD36 pmAB lacf‘ZAMlS] JMEa34-l Sm', JM 109 containing pEa34 This study JMUCDBOOI Km', JMlO9 containing pUCDBfX) This study JMGEM3 Amp', JM109 containing pGE‘M3Zf(+) This study Pseudomonas syringae Sm', containing pCPPSOI, isolated in New York 2, 27 pv. papulans Psp36 Other gram-negative Sm', various genera 36 bacteria (147 strains)” Plasmids pCPP505 A 2.1-kb fragment with and and strB from pCPPSOI cloned on pBR322 27 pEa34 Containing Tn5393 with strA and strB This study pGEMTN-l Tn5393 inserted on nucleotide 347 of pGEM3Zf(+) This study pGEMTN-7 Tn5393 inserted on nucleotide 331 of pGEM3Zf(+) This study pGEM3Zf(+) 3.2 kb with Amp' gene Promega Corp. pSTRBS A 2.5-kb BamHl-Sall fragment with $074 and strB from pCPP505 cloned This study on pGEM3lf(+) pUCD800 14.5 kb with Km' gene 8 ‘ Sm', streptomycin resistance; Sm’, streptomycin sensitivity; Km', kanamycin resistance; Amp', ampicillin resustance. All Sm' strains contained DNA that hybridized with probe SMP3. " One hundred forty of these strains contained DNA that hybridized with probe SAC32; five strains also contained DNA that hybridized with a 25.6-kb Smal fragment from pEa34. mid pEa34 in the transconjugants was confirmed by the method of Kado and Liu (15). Plasmid pEa34 was isolated and purified from transcon- jugant E. coli J MEa34-1 by alkaline lysis extraction followed by ccntrifugation an CsCl gradients (21). The plasmid was digested with restriction enzymes Apal, BamHI, NotI, Pstl, Sacl, Smal, and Xbal, and the sizes of restriction fragments were estimated by coclectrophoresis with l-kb DNA ladder markers (GIBCO-BRL, Grand Island, N.Y.). Restriction fragments were cloned into vector pGEM3Zf(+) to investi- gate the location of the streptomycin resistance genes. Detection of a transposon from pEa34. To test whether the determinant for streptomycin resistance on pEa34 was trans- posable, high-copy-number plasmids pGEM3Zf(+) and pUCD800 were used as recipient replicons to cntrap the element. Plasmid pEa34 was introduced into E. coli JM- GEM3 and JMUCD800 by conjugation from E. amylovom CA11 or E. coli JMEa34-1. Twenty-four transconjugant colonies were chosen randomly from each combination and grown in 5 ml of LB medium with streptomycin and either ampicillin or kanamycin for 16 h at 37°C and then subjected to plasmid analysis (15). Plasmid DNA from each transcon- jugant clone was isolated by alkaline lysis (21) and then transformed into competent E. coli JM109 cells (26). Trans- formants were selected by plating onto LB medium supple- mented with ampicillin or kanamycin, followed by replica plating of the resultant colonies onto LB medium supple- mented with the appropriate antibiotic plus streptomycin. Colonies resistant to streptomycin and either ampicillin or kanamycin and containing a plasmid only about 6.7 kb larger than the recipient replicons were selected for restriction enzyme analysis. Restriction mapping with Sacl, Aval. Pstl, and Hindlll and hybridization with radiolabcled pEa34 DNA were used to determine which portion of pEa34 had inserted into the plasmids and the insertion sites. Cloning and sequencing of DNA. Plasmids pSTRBS, pGEMTN-l, and pGEMTN-7 were used as the source DNA for nucleotide sequencing. Plasmid pSTRBS was con- structed by cloning a 2.5-kb BamHI-Sall DNA fragment of pCPP505 (27) in vector pGEM3Zf(+). Plasmids pGEMTN-l and pGEMTN-7 resulted from the capture of transposon Tn5393 in opposite directions on nucleotides 347 and 331, respectively, of pGEM3Zf(+). Deletions of the plasmids were performed with Exolll (Erase-a-Base kit; Promega Corp., Madison, Wis.). A nested series of deletion subclones of 200 to 250 bp each was selected, and their sequences were - determined by the dideoxy chain-termination method using denatured plasmid templates (11. 31). Nucleotide dGTP was replaced by dITP in some sequencing reactions to reduce band compression of the nucleotide sequence (Sequcnasc version 2.0, United States Biochemical). All sequences were confirmed by sequencing both strands from at least two overlapping clones. The nucleotide sequences and the de- duced amino acid sequences were analyzed with software from the University of Wisconsin Genetics Computer Group (UWGCG software, version 7.0; 5). Detection of Tn5393 and pEa34 in other gram-negative bacteria. A 3.2-kb SacI fragment from Tn5393, designated SAC32 (Fig. 18), was used as a probe in colony and Southern hybridizations to detect transposon Tn5393. Col- ony hybridization was used to screen 147 strains of strepto- mycin-resistant epiphytic gram-negative bacteria, which contained DNA that previously had hybridized with probe SMP3 (36). Southern hybridization was conducted to ana- lyze SacI-digestcd plasmid DNAs from P. syringae pv. papulans Psp36, E. amylovora ELOl and CA11, eight strains 34 734 CHIOU AND JONES A smut smt ruI Noll 33.7 33.9 34.0 2.4 ha] Sac] PslI B _. _ E .2. :3;";;:::- t‘ : aee§eecca5 s5 2° I I 11 III I 1 I Ir 1 I . I 6 I 2 3 i s 6' 670.5111: IR trIpA res InpR 151133 ser W3 IR <————-———> ——>—> probe probe SAC32 D—S—Mfl—s RSFIOIO m— D____ PP505 FIG 1. (A) lRestriction enzyme map of plasmid pEa34 fr ram E. amylovora .Eac restriction site is designated on the basis of its distance (in lkilabases) from the unique BamHI site. The location of streptomycin resistance genes str/1 and strB is indicated with a double arrow, and the location of transposon Tn5393 is indicated with a thick line. (B) The proposed genetic map for transposon TILS393. The proposed elements In Tn5393 are IR, transposase gene (mp/1), region res, resolvase gene (rnpR), insertion element (181133), str/1 and srrB genes, and IR, respectively. The directions of transcription of the genes are indicated with arrows. tians of probe eSMP3 fro rnP. syringae pv. papulans Psp36 eand of probe SAC32 are shown by the open boxes immediately below the genetic map. The locations of DNA sequences from RSFIOIO and P505 that are identical to the sequence in Tn5393 are shown b the slashed boxes, and the IR sequences are shown by the filled boxes. of the epiphytic bacteria which hybridized to SAC32 in colony hybridization, and two strains that did not hybridize. To detect plasmid pE334, a 25.6-kb Smal fragment from pEa34 was used as a probe in colony hybridizations with E. amylavom EIJ)1 and the 147 strains mentioned above. Plasmid pEa34 was used as the probe in Southern analysis of AMI-digested plasmid DNAs from six selected strains of Erwinia herbieola. Nucleotideseq uence accession number. The GenBank ac- cession number for the 6,705-hp transposon sequence is .1. BAC‘I'ERIOL. RESULTS Restriction map of pEa34. Plasmid pEa34 was transferred from E. amylovora CA11 into E. coli .1le by conjugation; plasmid DNA was isolated, and its restriction mapw determined. The unique BamHI site was used as a reference point for the restriction map of plasmid pEa34 (Fi g.1A.) The plasmid also contained unique Apal, Natl, and XbaI sites; three Sacl sites; three SmaI sites; and five Pstl sites. 0n the basis of the size of the restriction fragments, the plasmid was determined to be 34 kb in size. The smallest restriction fragment to confer resistance to streptomycin when ligated into plasmid pGEM3Zf(+) was located between the Xbal and Psrl (4.7) sites and was 2.8 kb in size. Detection of a transposable element from pEa34. When plasmid DNAs from 24 transconjugant colonies with pGEM3Zf(+) as the recipient replicon were examined, cells from 12 colonies contained a 9.9-kb plasmid that was not detected in the donor or recipient strains (data not shown). Cells of E. coli JM109 transformed with the 9.9-kb plasmid were resistant to both streptomycin and ampicillin. The high-copy-number plasmid was about 6.7 kb larger than the recipient replicon. Restriction mapping and Southern analy- sis of 9.9-kb plasmids from several transformants revealed that a 6.7-kb fragment from pEa34 had inserted into diflerent sites in pGEM3Zf(+). Similar results were obtained when plasmid DNAs from 24 transconjugant colonies with UCD800 as the recipient replicon were examined. The 6.7-kb fragment from pEa34 was considered to be a trans- posable element and was designated transposon Tn5393. Sequence andc coding regions of transposon Tn5393. The nucleotide sequence of Tn5393 p(F ig 2) was analyzed to produce a restriction enzymem and proposed genetic map for the transposon (Fig. 18). A asearch of the GenBank data base indicated that Tn5393 contained four functional open reading frames (ORFs) located between the 81-bp inverted repeats. The size and orientation of ORFs C and D suggested that ORF C encodes a transposase (tnpA) and ORF D encodes a resolvase (tnpR). ORF C encodes a polypeptide of 961 amino acids, is transcribed in a direction opposite to that of the other ORFs, and is terminated by a stop codon within the inverted repeat (IR). When the data base was searched for a putative polypeptide encoded by ORF C, identity of 29, 40, 31, 38, and 32% and similarity of 51, 61, 54, 57, and 56% with transposases of Tn3, Tn21, Tn9l 7, Tn2501, and Tn4430, respectively, were found (1, 12, 20, 41, 42). ORF D encodes a polypeptide of 181 amino acids. It exhibits 34, 34, 36 and 37% identity and 56, 52, 60, and 56% similarity with re- solvases of Tn3, Tn21, Tn917, and Tn2501, respectively (6, 12, 23, 35). However, ORF D revealed higher homology to integrascs of E. coli and bacteriophage Mu and recombi- nascs of Shigella boydii and bacteriophage P1 with 48, 47, 47, and 44% identity and 65, 63, 63, and 62% similarity, respectively (13, 28, 29, 40). A stretch of 194 hp, with two putative promoter sequences (nucleotides 2945 to 2940 and 3057 to 3062) for tIIIJe transcription of mpA and mpR, sepa- rates ORFs C and ORFs A and B encoded polypeptides of 267 and 278 amino acids, respectively. Computer aided sequence comparisons indicated that ORFs A and B share DNA sequence identity with ORFs H and I of plasmid RSF1010. The nucleotide sequence of ORF A differed by 3 bp (nucleotides 5163, 5426, and 5429) from the sequence of ORF H, and the sequence of ORF B was identical to the sequence of ORF 1 in RSF1010. These ORFs were designated strA and 508 by Scholz ct al. (32). A 1.2-kb sequence (nucleotides 3660 to 4891) with 27-bp 35 VOL. 175, 1993 NUCLEOTIDE SEQUENCE ANALYSIS OF T05393 735 a s. a .C ITCCGAGCGGCGAAACATGG 100 T L N I H E H G L P S V H lit 3 1 7 £4 . I I; '0... I C a 5 U .C ' P K P w R Y E G GCCAAGAGATCGGGCGATAGCAGCTTTCCATCGCGTTTCTCCTTTGCAACGACCTCGCCGAGCTTCATGGTGTTCCAGAAGATGATGATGGCCGCGAGCA 200 A L L D P S L L K G D R K Q N A V V E G L K M T N H F I I I A A L GATTCATGCCGGCGATGCGGTAATGCTGGCCTTCGGCGGAACGGTCGCGGATTTCACCGCGGCGGTGGAAGCTGATTGCCCGCTTCAGCGCATGATGACG 300 L N M G A I R Y H Q G E A S R D R I E G R R H F S I A R K L A H H R TTCGCCTTTGTTGAGCCCGATCTGGGCACGCCGTTGGAGTTCGGCATCCAGAATCCAGTCGATCATGAACAGGGTGCGCTCGACGCGACCGACTTCCCGC 400 E G K N L G I Q A R R Q L E A D L I H D I M F L T R E V R G V E R AGGGCTCTCGCGAGCTCGTTCTGCCGCGGATAGGAGGCGAGTTTCCGCAGAATCTGGCTTGGCGCGACGGTCCCGGCAGCAATGGTGGCGGCGATGCGCA 500 L A T A L E N Q R P Y S A L K R L I Q S P A V T G A A I T A A I R GGATGTCCGGCCAATTGCGCTCGATCATGGCTTGGTTGACCTTTCCGCCGATCAACGCTCGCAGGTGCGCCCGGGCGGCCGACGGATTGAACCCGTAGAG 600 L I D P W N R E I M A Q N V K G G I L A R L H A P A A S P N F A Y L CCGTTTGGATGGCACGTCGCGGATGCGCGGAGCCAACCGGTAGCCGAGAATGGCACATGCGGCAAACACGTGATCGGTCAACCCGCCCGTGTCCGTGAAC 700 R K S P L D R I R P A F R Y C L I A C A A F V H D T F G G T D T F TGCTCGCGGATATGGCGTCCAGCATCGTTCATCAGCAGGCCATCGAGGATGTAAGGCGCTTCGCTTGCCGTTGCAGGAATCACCTGGGTTGCGAACGGCG 800 Q E R I H R G A D N M L L G D L I Y P A E S A T A P I V Q T A F P CATATTGGTCGGAGACGTGGCTATAGGCTTTCAGGCCCCGGGTATTGCCATATTTCGCGTTGACCAGGTTCATGGCCTCACCTTGCTCTGTAGCGACGAA 900 A Y Q D S V H S Y A K L G P T N G Y K A N V L N M A E C Q E T A V F GAACTGTCCGTCGCTCGAAGCCGACGTGCCCATGCCCCAGAACCGGGCCATGGGTAACGCTGCCTGTGCCTCGACCACCATGGCCAGCGCCCGGTCATAG 1000 F Q G D S S A S T G M G W F R A M P L A A Q A E V V M A L A R D Y CCTTCGCCCTCGACATGCCACCGTCCAATGCGGATCAATTCCCAGAAGGTGTGGGTGTTTCTCCGATCCGCCATTTTGCGCAAGCCCAGGTTCATCCCTT 1100 A E G E V H H R G I R I L E H F T H T N T R D A M K R L C L N I G CCGCCAAGATAACGTTCATTAGCCCGATCCGGTCAGCGCACGGTGCTCCTGTGCCCAGATGGGTGAACGCTTCGGTGAAGCCGGTCGCCGCATCCACCTC 1200 E A L I V N M L G I R D A C P A G T R L H T F A E T F G T A A D V E CAGCAGGAGAILGGVGAPGLGLGlGGGLGGGATCTGCTTGTAGAGATCGAGCACCAGATCTTCGGCGCCTGTCGGCGCGGCGGCTTCGAGTTTCTCGATA 1300 L L L D T I R T P P I O K Y L D L V L D E A G T P A A A E L K H L TGCAGAACCCCGTTTTCAATCCACCCGCCCGGCATCGTGCCTGCGCGAGCGGCACCGCCAACCTCGCGCAACCGCATCTCGAGGCGAGCTTGCCGGTCTG 1400 V G N E I S G G P I T G A R A A R G V D R L R M D L R A Q R D E I CCAGCCATTCCTCCGCCCGCAATGGCACAGCGAGACGACCCCCTTCCGCGATGGATTGTGCCGGAACGAGTGCCTGTTTCAGATCGCCATAGCGCCGGGA 1500 A L W E E P R L P V A L R G G E A I S Q A P V L A H K L D G Y R R S CCTAGTAAGCCAGACATCTCCGGAGCGGAACGCATCGCGCAGATGGAACAGCACCGCGATCTCCCATAGGCGAGCGTCGCCAGCCCTCTGGGCCCGAAGG 1600 R T L W V D G S R F A D R L H F L V A I E W L R A D G A R Q A R L TGGCGATGCCATTTCCACCTGGGCCGCAAGAAGCTGGTCATCGCGGCATCGTTCAAACCCCTACGAACCCCCCTCACCCCTTCCAGAACCGGCAGTCCAA 1700 H R H W K S S P R L F S T M A A D N L G T R L A T V A E L L P L A CCGGCGCAGCTCGCAGATCGAGCAGGCGCAACATGCGTGGAGCGTATCGGCGGAAGCGGTGATAACCGTCGAGCACATGATTGAGCGGATCGTCGGCCAT 1800 V P A A R L D L L R L M R P A Y R R F R H Y C D L V H N L P D D A M GGTCGCGGTCAGCCTGGTTGCCATTGCAACAAGGGTTTTTAAGCCGTCCCACCCTCACCCACTCGCGATGACATCGCCCAGCCGCTCGCCATCATCCTGT 1900 T A T L R T A M A V L T K L G D W G S G S A I V D G L P Q G D D Q CCATCGACCAGGGCGCCCCCGATCTCGGCGAACCATTTCAGGCTGTCACCCACCACCCCCGCTTCGTCTGCGACCTTTCCATGGCAAATACGCTCCGAAG 2000 A D V L A G G I E A F S K L T D R V V G A E D A V K A H C I R E S CACGGTAGAGACGGCCGACGATCCGGTCGTGGGTTTCGACCACTGCGTCGGCCAACATCGCCTGCCATTCCGAGACGCAAACAGCCAAGATCGCAAGCCG 2100 A R Y L R G V I R D H T E V V A D A L M A O W E S V C V A L I A L R CCTGTCCTCCGGGAGATCGCGCATGCCGTCGGCATAATALLbtILALLQFGLLtGLGLAGACGAGTCACCCGATGGGCAGGAACGCCGGCAAGCAGATCC 2200 R D E P L D R M G D A Y Y R E C Q R R L R T V R H A P V G A L L D TCGGGGAGATCGATGCGTTGCAGATATTCGAGCCGGTCGAGCAGCCCGTTGGCCGACCAAGACTTCGAGCCAGGCTCGAACTCGCCCAGCCACACAAAAC 2300 E P L D I R Q L Y E L R D L L R N A S S S N S G P E F Q R L H V F GGGTCACCCGATCATCAGCCGTCTCCTCCAGCAATGCCAGCAACTGTTCTCGGATCGACATACGCAGCCCACTGGCGATCCTCGTCTCGATGCGTCGCTC 2400 R T V R D D A T E E L L A L L Q E R I S M P L R S A I R T E I R R E GGCATCGACGAGAGCCGCGGCACAAAGCCGCTCGATCGTGGATGTCGCGCGAAGGACAGTGCGCGTGCGTCGGCACTCGGCTACGAAGCGACGGGCGATA 2500 A D V L A A A C L R E I T S T A P L V T R T R R C E A V F R R A I TCCTCGTTCGACACCCCCATCTCCGCTTCTCGGAACAACCATTCCTTCAGCTCGCTCGCACCACCTCCGCAGAAGGTGCGGAAGCCGTAGAGCCCCCCTA 2600 D E N S V A M E A E R F L W E K L E S A G R G S F T R F G Y L C R ACTCGGCAACATGCTCGTGCCGTGTTTCCTCGCGGGCAGCATAGTCTACGAGATCGTCGCCACCCAGGCCAAGCTGCGCTCCGATAAATTCGATGACCTC 2700 L E A L H E H R T E E R A A Y D V L D D A G L G L Q A G I F E I V E TGCAGGCATCAGTTCGCCTGGAGCCAGCACCCGGCCGGGATAGCGCAGGACACACAATTGCAGGGCGAAGCCGAACCTCTTGTCAGCGCCCCGACGCACC 2800 A P I L E C P A L V R G P Y R L V C L Q L A F G F R N H A R R R L FIG. 2. 36 736 CHIOU AND JONES J. BACTERIOL. CTGATATGCCCAAGGTCTTCATCACTCAGCGTATAGTGCTTGAGCAAATCCCTCTCTGAAGTCGGCAAGCGCAACAGCGCGTCTTTCTCCCGATCGGTTA 2900 R I H G L D E D S L T Y H K L L D T Q S T P L R L L A D K Q R D T -10 -35 GAGTGACGCGACGCGGCATACATCTTCCTTTTTCAAAATCTCATAGCGTTCAAGACCCTTTGTTTATGAAGCTGGTTGAGATACATTTCCACAGGTCAAT 3000 L T V R R P M --- ORF C -35 -10 GCAATCGTGGCCGAAGCGCCGCCTCAAACCAACGTTTGTGATACATGCTGATCGGATATGCCCGCGTCTCCAAAGCCGATGGCTCGCAGTCTCTCGACCT 3100 GCAGCACGACGCCTTGCGCGCCGCAGGTGTCGAACGGGACAATATCTATGATGATCTTGCTTCCGGCGGTCCTGATGATCGCCCTGGCTTGACTGCCTGC 3200 ORF D --- H R A A G V E R D N I Y D D L A S G G R D D R P G L T A C CTCAAGTCATTGCGTGACGGCGATCTGCTGGTGGTCTGGAAGCTCGATCGCCTCGGACGATCGCTTGCCCATCTGGTCAACACGGTGAAGGAGCTGTCAG 3300 L K S L R D G D V L V V W K L D R L G R S L A H L V N T V K E L S D ACCGCAAGATCGGCCTGCGGGTTCTGACTCGAAAGGGCGCTCAGATCGACACCACGACTCCGTCCGGTCGCATGGTGTTCGGAATCTTCGCCACCTTGGC 3400 R K I G L R V L T G K G A Q I D T T T A S G R M V F G I F A T L A CGAGTTCGAGCGGGATCTGATCCGAGAGCGCACCATGGCGGGTCTCGCCTCCGCGAGAGCGCGCGGTCGCAAGCGCGGACGAAAATTCGCGCTCACCAAA 3500 E F E R D L I R E R T M A G L A S A R A R G R K G G R K F A L T K GCTCAGGTGCGTCTCGCGCAAGCCGCCATGGCCCAGCGCCATACTTCAGTTTCCGATCTCTGCAAGGAACTCGGCATCGAGCGCGTCACTCTCTACCGAT 3600 A Q V R L A Q A A M A Q R D T S V S D L C K E L G I E R V T L Y R Y ATcchc'rcccAAAGGCGAGCTCAGAGACCA't‘ccAAAGCATGTTCTCGGACTTACGWTTTTTGAGAGC ATT 3 7 oo VGPKGELRDHGKHVLGLT' ATTTGTTATCAAGGAGACCATATATGTCATTAAAGCATAGTGATGAATTTAAGCGTGATGCAGTTCGCATAGCACTCACTAGTGGCTTAACACGCCGTCA 3800 AGTTGCGTCAGATTTAAGTATTGGGCTTTCCACGCTTGGGAAATGGATCGCATCAATTTCCGATGAAACTAAAATTCCTACCCAAGACACTGATCTTCTG 3900 CGTGAGAATGAACGTTTACOCAAAGAGAACCGTATCCTTCGGGAGGAGAGGGAGATATTAAAAAAGGCAGCAATATTTTTCGCAGTACAAAAGCTGTGAG 4000 ATTTCAGTTTATTACGGATTACCGTGGCTCTCTCTCACGTTCACCCATATGTCGTTTGATGGGCGTAACAGATCGTCGTTTACGTGCATGGAAACGCCGT 4100 CCTCCATCACTGCGCCAGCGTCGTGATCTTATACTTCTAGCGCATATACGTGAGCAGCATCGGTTCTGTTTGGGGAGCTATGGTAGGCCGCGTATGACAG 4200 AAGAGTTGAAAGCGCTGGGCCTGCAGGTTGGGCAGCCTCGGGTTGGACGTTTGATGCGCCAGAATAACATTACAGTTGTTCGAACGCGTAAATTCAAACG 4300 GACAACGGATAGTCATCATACCTTCAACATTGCACCGAACCTATTAAAACAAGACTTTAGCGCAAGCGCACCCAACCAGAAATGGGCAGCCGATATCACT 4400 TATGTTTGGACCAGAGAAGGATGGG1LfAlLItbLtblIATCCTTGACCTGTATTCCCGTCGCGTGATTGGCTCGGCAACAGGTGATCGATTAAAGCAGG 4500 ATCTTGCATTAAGGGCACTGAATATGGCGTTGGCTTTACGCAAACCACCACCGCGTTGTATTCAACACACACACCGTGGGAGCCAATATTGCGCTCATGA 4600 ATATCAAAAGCTACTGCTCAAACATCAATTGCTGCCGTCCATGAGCGGGAAAGGCAATTGTTTTGATAACTCCCCAGTAGAAAGCTTCTTTAAATCATTA 4700 AAGGCTGAGTTGATTTGGCGCAGACACTGGCAAACAAGGCGAGATATTCAGATTGCAATCTTCGAATATATAAATGGCTTTTATAATCCACCCCGAAGAC 4800 -35 ~10 ATTCAACACTCGGCTGGAAATCGCCGGTGGCATTTCAGAAAAAAGCCGCTTAAAATGAGAGATAC C CC C CCCT C C 'CCAACTC 4900 GTTTCTTTTCGCAGGTTGAGCCACCTCCGCGCTTCATCAGAAAACTGAAGGAACCTCCATTGAATCGAACTAATATTTTTTTTGGTGAATCGCATTCTGA 5000 ORF A --- M N R T N I F F G E S H S D CTGGTTGCCTGTCAGAGGCGGACAA1LtbbtbAtt11bittttLCACGTGGTGACGGGCATGCCTTCGCGAAAATCGCACCTGCTTCCCGCCGCGGTGAG 5100 w L P V R G G E S G D F V F R R G D G H A F A K I A P A S R R G E C CTCGCTGGAGAGCGTGACCGCCTCATTTGGCTCAAAGGTCGAGGTGTGGCTTGCCCCGAGGTGATCAACTGGCAGGAGGAACAGGAGGGTCCATGCTTGG 5200 L A G E R D R L I W L K G R G V A C P E V I N W O E E Q E G A C L V TCATAACGGCAATTCCGGGAGTACCGGCGCCTGATCTGTCTGGAGCGGATTTGCTCAAAGCGTGGCCGTCAATGGGGCAGCAACTTCGCGCTGTTCACAG 5300 I T A I P G V P A A D L S G A D L L K A W P S M C Q Q L G A V H S CCTATCGGTTGATCAATGTCCGTTTGAGCGCAGGCTCTCCCGAATGTTCGGACGCGCCGTTGATCTGGTGTCCCGCAATGCCGTCAATCCCGACTTCTTA 5400 L S V D Q C P F E R R L S R M F G R A V D V V S R N A V N P D F L CCGGACGACGACAAGAGTACGCCGCAGCTCGATCTTTTGGCTCGTGTCGAACGAGAGCTACCGGTGCGGCTCGACCAAGAGCGCACCGATATGGTTGTTT 5500 P D E D K S T P Q L D L L A R V E R E L P V R L D Q E R T D M V V C L H CCCATGGTCATCCCTGCATGCCGAACTTCATGGTGGACCCTAAAACTCTTC TGCACGGCTCTGATCGACCTTCGGCGGCTCGGAACAGCAGATCGCTA 5600 H G D P C M P N F M V D P K T L Q C T G L I D L C R L G T A D R Y TGCCGATTTGGCACTCATCATTGCTAACGCCGAAGAGAACTGGGCAGCGCCAGATGAAGCAGAGCCCGCCTTCGCTGTCCTATTCAATCTATTGGGGATC 5700 A D L A L M I A N A E E N W A A P D E A E R A F A V L F N V L G I CAAGCCCCCGACCCCGAACGCCTTGCCTTCTATCTGCGATTGGACCCTCTGACTTGGGGTTGATGTTCATGCCGCCTGTTTTTCCTGCTCATTGGCACGT 5800 E A P D R E R L A F Y L R L D P L T W G ' ORF B --- H F M P P V F P A H w H V FIG . 2—Continucd. 37 VOL. 175, 1993 NUCLEOTIDE SEQUENCE ANALYSIS OF Tn5393 737 IILGLAALLIGI1LILATTGCCGACACCTTTTCCAGCCTCGTTTGGAAAGTTTCATTGCCAGACGGCACTCCTGCAATCGTCAAGGGATTGAAACCTATA S900 S Q P V L I A D T F S S L V H K V S L P D G T P A I V K G L K P I GAAGACATTGCTGATGAACTGCGCGGGGCCGACTATCTGGTATGGCGCAATGGGAGGGGAGCAGTCCGGTTGCTCGCTCGTGAGAACAATCTGATGTTGC 6000 E D I A D E L R G A D L V W R N G R G A V R L L G R _E N N L M L L TCGAATATCCCGGGGAGCGAATGCTCTCTCACATCGTTGCCCAGCACGGCGACTACCAGGCGACCGAAATTGCAGCGCAACTAATGGCGAAGCTGTATGC 6100 E Y A G E R M L S H I V A E H G D Y Q A T E I A A E L M A K L Y A CGCATCTGAGGAACCCCTGCCTTCTGCCCTTCTCCCGATCCGCGATCGCTTTGCAGCTTTGTTTCAGCGGGCGCGCGATGATCAAAACGCAGGTTGTCAA 6200 A S E E P L P S A L L P I R D R F A A L F Q R A R D D Q N A G C Q ACTGACTACGTCCACGCGGCGATTATAGCCGATCAAATGATGAGCAATGCCTCGGAACTGCGTGGGCTACATGGCGATCTGCATCATGAAAACATCATGT 6300 T D Y V H A A I I A D Q M M S N A S E L R G L H G D L H H E N I M F TCTCCAGTCGCGGCTGGCTGGTGATAGATCCCGTCGGTCTGGTCGGTGAAGTGGGCTTTGGCGCCGCCAATATGTTCTACGATCCCGCTGACAGAGACGA 6400 S S R G W L V I D P V G L V G E V G F G A A N M F Y D P A D R D D CCTTTGTCTCGATCCTAGACGCATTGCACAGATCGCGCACGCATTCTCTCGTGCGCTCGACGTCGATCCGCGTCGCCTGCTCGACCAGGCGTACGCTTAT 6500 L C L D P R R I A Q M A D A F S R A L D V D P R R L L D Q A Y A Y GGGTGCCTTTCCGCAGCTTGGAACGCGGATGGAGAAGAGGAGCAACGCGATCTAGCTATCCCGGCCGCGATCAAGCAGGTGCGACAGACGTCATACTAGA 6600 G C L S A A W N A D G E E E Q R D L A I A A A I K Q V R Q T S Y 6705 TATCMGCGACNCNCTATCCCCWWW 5 7 0 0 aces: FIG. 2. Complete nucleotide sequence of Tn5393, numbered from the IR on the tnpA end, is shown in 5’ to 3' orientation. The positions of ORFs corresponding to four predicted polypeptides (A, B, C, and D) are indicated with their encoded amino acid sequence. ORF C is transcribed from the strand opposite to ORFs A, B, and D. The nucleotide sequence of insertion sequence 181133 is from nucleotides 3660 to 4891. The 3-bp direct repeats that border 181133 (TAG) are double underlined, and the IRs of Tn5393 and 181133 are underlined. The -10 and -35 consensus sequences of three putative promoters are indicated above the sequence by a line and the number. Asterisks indicate stop codons. The nucleotide and amino acid sequences of ORF A that differ from those of ORF I in RSFIOIO are indicated above the nucleotide and beneath the amino acid, respectively. Sequence 4928 to 6705 of Tn5393 is homologous to sequence 31 to 1808 of RSFIOIO (32). IRs and 3-bp duplicates separated tnpR and srrA and was designated insertion element 181133. Its nucleotide sequence shared 45% identity with insertion sequence [83 (39). 181133 provided a promoter (nucleotides 4851 to 4856) for transcrip- tion of the resistance genes. When nucleotides were deleted from pGEMTN-7, streptomycin resistance was lost when deletions were made downstream, but not when deletions were made upstream, of the promoter sequence (data not shown). The 81-bp IRs of Tn5393 were about twice the length of IRS typically found in Tn3-typc transposons. The sequences of the two IRs (IR-53930 and lR-5393t) differed by 4 bp (Fig. 3A), and both IRs were flanked by S-bp direct repeats. The stop codon for ORF C was located in nucleotide positions 34 to 36 of IR—5393t. A stop codon was also found in the IRs of transposons Tn3, Tn21, and Tn2501, but not in the IRS of transposon Tn917 or Tn4430 (Fig. BB). The sequence of lR-5393t differed by 1 bp, and the sequence of IR-53930 differed by 4 bp, from nucleotides 1728 to 1808 in plasmid RSFIOIO (32; Fig. 3A). Identity of streptomycin resistance in P. syringae pv. papu- lans and E. amylovom. The nucleotide sequence of the 2.5-kb BamI-II-Sall fragment from pCPP505, a DNA clone from P. syringae pv. papulans Psp36 that confers resistance to streptomycin in E. coli (27), was compared with the se- quence of Tn5393. The first 241 nucleotides in the DNA fragment from pCPP505 were identical to nucleotides 3419 to 3659 in tnpR of Tn5393, and nucleotides 242 to 2052 were identical to the sequence for the stud and strB genes of Tn5393. The nucleotide sequence of 181133 and a direct repeat (TGA) in Tn5393 were absent in the fragment from pCPP505. Fragment SMP3 from P. syringae pv. papulans (27), which has been used as a probe for the detection of streptomycin resistance genes in many gram-negative bacte- ria (27, 36), consists of sequences from the tnpR and SIM genes of Tn5393 (Fig. 18). Tn5393 in other gram-negative bacteria. In colony hybrid- izations, probe SAC32 hybridized with DNA from 140 of 147 epiphytic gram-negative bacteria from the study of 80bi- czewski et al. (36). In Southern hybridizations, the probe hybridized with a 3.2-kb Sacl restriction fragment from P. syringae pv. papulans Psp36, E. amylovora CA11, and eight strains of epiphytic bacteria, which reacted positively to SAC32 in colony hybridizations. However, the fragment was 10 20 30 40 50 “2-53930 GGGG‘I‘CGT‘I'I‘ GCGGCAGACG GCGAAA'T‘CCT ACGCTAAGGC TT'I'SGCCAAC IR-5393t GGSG’I‘CS'IY'T GCSGGAGOJS GCGGf-JITCCT ACCCTAAGGC TTTCGCCAGC RSFIOIO GGCIGTCG'IT'I' GCGGGAAGGG GCGGAATCCT ACGCTAAGGC WCAOC 60 70 80 IR- 53930 GATAl'l'LTCC GGTAAGA'.'I‘C AT‘STCI'I'CCC A IR - 53 93C GATE ."I'i ICC C-C’TOAGA .".‘G ATST‘GT'IL‘CC A RSFIOIO DATA I'ICTCC GG'TGAGAI'IIJ ATUTGI'I'CCC A B. 10 20 30 40 IR‘5393t GGGGTCGT’I'I' GCGGGAGGG-S GCCGAAT'CCT ACGCIAACGC TT'TGGCCA' ' '- IR - 3 t GGGGTCT‘GAC GL‘T'CAGT‘SGA AC'JI'MACTC ACGI‘IEyRG l2 2 l LR 21t 633370,; I‘CT CA'JAAAACGG Alck.-'«T.WIGC AC Fifi-AG l 2 2 ) IR 917C GG-SST‘CCCGA CCI‘JCT'IA-CTG GOA/\mSTA “FCC-ATM? (19) IR-2501t GC‘v-SG'DCCGCC 'I‘CCAAARCGG AAATTATCCC ACCCJZMGAC TC'I‘T'I'I'I'I' (221 IR'4430t GGGGTACCGC CAGCATTTCG GAAAAAAACC ACGCTAAG (191 FIG. 3. Comparison of IR sequences. (A) The 81«bp IRs of Tn5393 (IR-5393t and IR-5393o, where t is the tnpA end and o is the other end) and the sequence of plasmid RSF1010. Boldface type shows positions where residues were different. (B) IRS in represen- tative transposons from the Tn} family (Tn5393, Tn3, Tn21. Tn917, Tn2501, and Tn4430) are all listed from the rnpA end. The stop codons for the transposases in Tn5393, Tn3, Tn21, and Tn2501 are underlined. The number in parentheses is the number of identical base pairs between each IR and IR-5393t. Sequences are from Scholz et al. (32) for RSFIOIO, Heffron et al. (12) for Tn3, Zheng et al. (45) for Tn21, An and Clewell (l) for Tn917, Michiels and Cornelis (22) for Tn2501, and Mahillon and Lereclus (20) for Tn4430. 738 CHIOU AND JONES 123.45678910111213 ' it t . ;2_.__ .. .- on.‘ i - r. . .. FIG. 4. Autoradiograph of a Southern blot of plasmid DNAs from streptomycin- -resistant (except lane 3) gram-negative bacteria digested with Sacl and hybridized to probe SAC32. Lane I, P. synngaepv. papulans Psp36; lane 2, E. nmylovom CA11; lane 3, E. amylovom £1131; lane 4, Acinetobacrer sp. strain 1; lane 5, E. herbicala 6a; lane 6, E. herbicala 34; lane 7, ayellow Pseudomonas strain,40; 1ane8, P.3'y syringae pv. syringae 45; 1ane9, Pseudomanas fluores enshiovar III, 57; lane 10,P.reudomona.raen4gt'nosa lane 11, P. purida 61, lane 12, Aeromonas sp. str rain 145; lane 13, E. herbicola 144. The size of the 3.2-kb hybridizing fragment is indicated at the far left. not detected in streptomycin- -sensitive E. amylovora EL01 and streptomycin- -resistant Pseudomonas purida 61 or Aer- omonas sp. strain 145 (Fig. Plasmid pEa34 in other gram- negative bacteria. In colony hybridizations, a 25. 6- kb SmaI fragment from pEa34 hybrid- ized with DNAs from five strains of E. herbicola and from streptomycin-sensitive E. amylovora EL01 but not with DNAs from 142 additional epiphytic gram-negative bacterial strains. When plasmid DNA was separated by agarose gel electrophoresis, the five strains of E. herbicola each con- tained a 34-kb plasmid, whereas E. amylovora EL01 con- tained a 28-kb plasmid. Among the five strains of E. herbi- cola, only three strains showed Aval restriction patterns identical to that of plasmid Ea34 in Southern blots when hybridized with radiolabcled pEa34 DNA (Fig. 5), indicating that the transposon inserted on difi‘erent sites of the proto- type plasmid. In conjugation studies, a 34-kb plasmid was transferred from E. herbicola 144 to E. coli JM109, JM- GEM3, and JMUCD800 and to E. amylovam BC06 in high frequency (data not shown). DISCUSSION We suggested in a previous paper that the plasmid pEa34 may have arisen from the Insertion of a transposable element into an existing plasmid or that E. amylovora may hav acquired a new plasmid (3). Except for streptomycin- resis- tant E. amylovora from Michigan, a 34-kb plasmid resem- bling pEa34 has not been reported among strains of E. amylavora examined for cryptic plasmids (7, 15, 37) Plas- mid pEa34 likely originated from the insertion of Tn5393 into a-28 kb plasmid; we detected a 28- kb plasmid with DNA homologous to pEa34 In streptomycin- -sensitive strain EL01 of E. amylovom. Hybridization studies indicated that the 28-kb plasmid and plasmid pEa34 were not related to the 29-kb universal plasmid found in pathogenic strains of E. amylovora (7, 18). Whether sensitive strains of E. herbicola 38 J. BACTERIOL. 1 234567 FIG. 5. Hybridization of a 3.6-kb Smal fragment from plasmid pEa34 with AMI-digested total plasmid DNAs from streptomycin- resistant strains lof E. herbrcola. Lanes 1 to 6 contain DNAs from 52, 50a, 44,35a, 18, and 6a, respectively. Lane 7 contains plasmid pEa34 from E. amylavara CA11. Strain 6a was a negative control. The threeAvaI fragments indicated (by size in kilobases) at the far left are internal fragments of Tn5393. A 127-bp fragment is not seen harbor a 28- kb plasmid homologous to the plasmid found In strain EL01 needs to be det ned We classify Tt15393 as a cclalss II, Tn} like transposon because it makes a 5-bp target duplication, and its trip! and tnpR are transcribed in opposite directions (17). A large number of antibiotic resistance transposons isolated from gram-negative bacteria associated with humans and animals are Tn3-like elements carried often by conjugative plasmids. In this study, we extend the host range of TILi-like trans- posons to gram- negative bacteria associated with plants. The detection of streptomycin resistance in many gram- negative conjugational transfer of the plasmid within a bacterial species and later between ecologically associated species. Tn3 transposons have been divided into five subgroups (9). The near-perfect 81-bp IRs at the ends of Tn5393 were much longer than the 384 to 45-bp IRs common to other transposons in this family (9), and amino acid sequence identity of transposases and rcsolvases between Tn5393 and other Tn3- like transposons was low (29 to 40%). Therefore, our results suggest that Tn5393 should belong to a new subgroupn In the Tn3 family The nucleotide sequences associated with streptomycin resistance in E. amylovom CA11 and P. syringae pv. papu- lans Psp36 were identical to the MM and srrB genes for streptomycin resistance in the enterobacterial. plasmid RSFIOIO (32). Streptomycin resistance in pv. syringae is also related on the basis of sequence analysis to the streptomycin genes in RSF1010 (38). Our sequence data confirmed that probe SMP3 is part of the streptomycin resistance gene from P. syringae pv. papulans (Fig. IB). Previously, DNA homologous with probe SMP3 was de- tected in many streptomycin-resistant gram-negative bacte- ria from plants (27, 36), and DNA similar to or homologous with the str/1 and srrB genes of RSFIOIO was detected in many streptomycin-resistant gram-negative bacteria of ani- mal, including human, origin (19, 30, 43). The finding that bacteria from plants have the streptomycin resistance genes 39 VOL. 175. 1993 found in bacteria from human and veterinary clinics extends the importance of this resistance determinant. Furthermore, RSFIOIO contains an 8l-bp sequence downstream of am and srrB that is homologous to the IRs of Tn5393, suggesting that the streptomycin resistance genes on RSFIOIO and Tn5393 are derived from a common ancestor. The transpo- sition of Tn5393 to various mobilizable plasmids could have played an important role in the widespread distribution of the streptomycin resistance genes among gram-negative bacteria. Restriction fragment length polymorphism was observed in Avai digests of plasmid DNAs from various epiphytic gram-negative bacteria isolated from apple orchards in New York and Michigan (27, 36). In most orchard bacteria, probe SMP3 hybridized with a single Aval fragment of either 2.7 or 1.5 kb (27, 36). We noted that the difference of about 1.2 kb corresponded to the size of insertion sequence 181133 and subsequently confirmed by restriction analysis that 181133 was present in the 2.7-Kb fragments but not in the 1.5-kb fragments (data not presented). The detection of two forms of transposon Tn5393 indicates that the resistance in E. amylovora and some strains of E. herbicola probably evolved independently, or at least that its evolution has diverged, from that in P. syringae pv. papulans and most other fluorescent pseudomonads. For bacteria in which probe SMP3 hybridized with a single Aval fragment smaller than 1.5 kb, DNAs from such bacteria often contained the SIM and srrB genes but not the whole tranSposon (data not shown). Evolution of streptomycin resistance in these bac- teria probably resembles that for plasmid RSF1010. We detected insertion sequence 181133 in transposon Tn5393 from streptomycin-resistant E. amylovora CA11. Although transposon Tn5393 was common in streptomycin- resistant bacteria isolated from apple orchards, it did not generally harbor 181133. However, a few strains of strepto- mycin-resistant E. herbicola harbored not only Tn5393 and 181133 but also a plasmid that hybridized with pEa34 from E. amylovora CA11. The presence of Tn5393 and 181133 in- serted in a plasmid common to two species of bacteria indicates a possible epidemiological association between E. amylovora and E. herbicola. ACKNOWLEDGMENTS This research was supported in part by the Michigan Apple Research Committee and the Michigan Agricultural Experiment Station. We thank Shauna C. Somerville for her comments on the manu- script. REFERENCES 1. An. F. Y., and D. B. Clewell. 1991. Tn917 transposase sequence correction reveals a single open reading frame corresponding to the MM determinant of Tn3-family elements. Plasmid 25:121- 124. 2. Burr, T. J., J. L. Norelli, B. Katz, W. F. Wilcox, and S. A. Hoylng. 1988. Streptomycin resistance of Pseudomonas syrin- gae pv. papulans in apple orchards and its association with a conjugative plasmid. Phyt0pathology 78:410-413. 3. Chiou, C.-S., and A. L. Jones. 1991. The analysis of plasmid- mediated streptomycin resistance in Erwinia amylovora. Phyto- pathology 81:710—714. 4. Coyier, D. L., and R. P. Covey. 1975. Tolerance of Erwinia amylovora to streptomycin sulfate in Oregon and Washington. Plant Dis. Rep. 59:849—852. 5. Deverenx, 1., P. Haeberli, and O. 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Characterization of a streptomycin-sulfonamide resistance plas- mid from Actinobacillus pleumpneumoru‘ae. Antimicrob. Agents Chemother. 33:235—238. . Yanisch-Perron, C., J. Vieira, and J. Measlng. 1985. Improved M13 phage cloning vectors and host strains: nucleotide se- quences of the M13mp18 and pUC19 vectors. Gene 33:103—119. Zheng, Z. R., M. Chandler, R. Hlpsldnd, M. Clerget, and 1... Cam. 1981. Dissection of the r-determinant of the plasmid R100.1: sequence at the extremities of Tn21. Nucleic Acids Res. 9:6265-6278. PART III IDENTIFICATION OF Two AMINOGLYCOSIDE PHOSPHOTRANSFERASES, APH(3")-lb AND APH(6)-Id, ENCODED BY THE aph(3")-Ib (strA) AND aph(6)-Id (strB) GENES FROM STREPTOMYCIN-RESISTANT Erwinia amyl ovora ABSTRACT The plasmid-borne strA and strB genes from Erwinia amylovora strain CA11 were characterized by genetic and biochemical analyses. In deletion experiments, deletions in strB resulted in a reduction in the minimum inhibitory concentration (MIC) from 500 to 100 pg streptomycin m1"1 and in strA from 500 to 25 pg streptomycin ml'1 or less. When strA and strB were cloned separately on a lacIQ/Ptac-based expression vector in EScherichia coli, the protein encoded by strA, but not the one encoded by strB, was overexpressed. Sequence analysis of the overlapping genes indicated that the distal strB gene lacked a Shine-Dalgarno sequence and that the initiation codon was in the double-stranded region of the stable stem-loop structure. Conversely, the Shine- Dalgarno sequence and the initiation codon in strA were exposed in the single-stranded loop of a stable stem-loop structure. The strB gene was overexpressed and resistance restored to a MIC of 100 pg streptomycin ml"1 by introducing a Shine-Dalgarno sequence and by altering the mRNA secondary structure. 13C-NMR analysis of the respective phosphorylated streptomycin products indicated that strA- strB encoded aminoglycoside-3"-phosphotransferase [APH(3")- Ib] and aminoglycoside-6-phosphotransferase [APH(6)-Id], 42 43 respectively. These data suggest that the high level of resistance to streptomycin exhibited by bacteria with strA- strB genes is due to the coexistence in the cells of APH(3")-1b and APH(6)-Id enzymes and that the differential expression of these enzymes is regulated by the mRNA secondary structure. INTRODUCTION The DNA associated with streptomycin resistance in strain CA11 of Erwinia amylovora is carried on transposon Tn5393 which is inserted on a 34-kb conjugative plasmid pEa34 (3, 4). The region of pEa34 affecting resistance consists of two open reading frames that have sequence identity with the strA and strB genes of plasmid RSF1010 (4, 10, 19). Hybridization studies indicate that these genes are also present in many gram-negative streptomycin—resistant bacteria isolated in Michigan and New York from the apple orchard environment (2, 16, 21). Streptomycin is a member of the aminoglycoside- aminocyclitol class of antibiotics. Aminoglycosides can be inactivated by phosphorylation, adenylylation, or acetylation. The enzymes reported to modify streptomycin include aminoglycoside-3"-phosphotransferase, APH(3")-I; aminoglycoside-6-phosphotransferase, APH(6)-I; aminoglycoside-3"-nucleotidyltransferase, ANT(3")-I; and aminoglycoside-G-nucleotidyltransferase, ANT(6)-I (5, 20). Based on sequence comparison analysis, Shaw et a1. (20) proposed that the strA—strB genes in RSF1010 encode APH(3")- Ib and APH(6)-Id, respectively. The identity of the enzymes encoded by strA-strB remains to be confirmed by biochemical 44 45 analysis. In this research, we analyzed the strA-strB genes from E. amylovora plasmid pEa34 by deletion analysis and investigated the role of the stem-loop structure around the ribosome-binding site on the translational regulation of strB. Finally, the enzymes encoded by strA and strB were identified. MATERIALS AND METHODS Bacterial strains, plasmids, and primers. The bacterial strains, plasmids, and primers used in this study are described in Table 1. E. coli JM109 (25) was routinely used as the host for plasmids and gene expression. Strains of E. coli were grown at 37°C on Luria-Bertani (LB) agar or in LB broth (13). Plasmid pMMBGGHE (7) was kindly supplied by M. Bagdasarian, Michigan State University, East Lansing. The primers were synthesized with an automatic 3808 DNA Synthesizer (Applied Biosystems, Foster City, CA) at the Macromolecular Facility Laboratory, Michigan State University. DNA manipulations and construction of expression vector pTWNHB. Plasmid preparations, DNA restriction digests, agarose gel electrophoresis, isolation of DNA fragments, ligations, and fill-in reactions were done using standard methods (13). A 4,018-bp multiple copy expression vector pTWNHE was constructed by ligation of a 1,550-bp ScaI-PvuII fragment from pGEMBZf(+)(25) and a 2,468-bp Eco47III-ScaI fragment from pMMBGéHE (7). The hybrid plasmid consisted of the replication origin of pGEM3Zf(+), a B-lactamase gene (bla), lacIQ, lac operator site, tac promoter, a polylinker, and two transcriptional terminators of the E. coli rrnB 46 47 TABLE 1. Bacterial strains, plasmids and primers Source or Designation Relevant characteristic(s) reference(s) Bacterial strains Escherichia coli JM109 endAI recAl gyrA96 thi hst17(rK' mx+ ) relAJ Promega Corp. supE44 1‘ A(lac-proAB) [F' traD36 proAB lac193 25 ZAMls] SMABBQ JM109 (pTWNHEzzstrA2839) This study SMBClS JM109 (pTWNHEzzstrB-C15) This study SMBEl JM109 (pTWNHE::strB-AJ24) This study SMBl JM109 (pTWNHE::strB-AJ25) This study SMBlO JM109 (pTWNHE::strB-AJ26) This study SMBlON JM109 (pTWNHE::strB-AJ26N) This study SMBA9 JM109 (pTWNHE::strB—AJ27) This study SMBD3 JM109 (pTWN ::strB-AJ28) This study SMBCS JM109 (pTWNHE::strB-AJ34) This study Plasmidsa pEa34 Smr, strA-strB, Tn5393, conjugative 4 pTWNHE Apr, a multiple copy expression vector derived from pMMBGGHE and pGEM32f(+) This study pGEM32f(+q.Apr, multiple copy plasmid vector Promega Corp. pMMBGGHE Apr, a broad-host-range expression vector derived from RSF1010 7 pSTRXP .A 2.8-kb XbaI-PstI fragment with strA-strB 48 Table 1 (cont'd) from pEa34 cloned on pGEM32f(+) This study pC2, pF16 Deletion derivatives of pSTRXP containing both intact strA and strB This study p321 Derivative of pSTRXP strA disrupted by removing a 272-bp NruI fragment This study pC8, pClS Deletion derivatives of pSTRXP with intact strB, disrupted strA This study pBS, p839 Deletion derivatives of pSTRXP with intact strA, disrupted strB This study pA17 Deletion derivative of pSTRXP with disrupted strA and disrupted strB This study Primersb AJ23 d(GGAGAAITCGCTTGATATCTAGTA) This study AJ24 d(CTCTAAGGAAGGGTTGATGTTCATG) This study AJZS d(IAAQQAQIIAAQQIIATGTTCATGCCGCCTG) This study A326 d(TAAGGAGGTTAACGTTATGTTCATGCCGCCTG) This study AJ27 d(IAAQQAQIIAAQQIIATGCCGCCTGTTTTTC) This study AJ28 d(WATGCCGCCTGTTTTTC) This study AJ3O d(ATGTGTGGAATTGTGAGCGG) This study AJ34 d(IAAGGAGGIAGGGTTGATGTTCATGCC) This study a Smr = streptomycin resistance; Apr = ampicillin resistance. b Nucleotides in each sequence that could pair with the 3' end of E. coli 168 rRNA (AUUCCUCCACU... 5') are shown in larger size print. Nucleotides not present in the original strB template are underlined. 49 operon from pMMBGGHE. Enzyme assays. APH enzyme activity was detected using a phosphocellulose paper binding assay (5). All bacteria were grown in LB broth at 37°C in a shaking incubator until the optical density at 600 nm was 0.6. Isopropyl-B-D- thiogalactopyranoside (IPTG) was then added (final concentration 0.5 mM) to the culture and shaking was continued for 4 h. Crude enzyme extracts were prepared by sonication (Vibra CellTM, Sonics & Materials Inc., Danbury, CT). Incubation of streptomycin and [y-P32]ATP with the sonicated enzyme extract, washing of the phosphocellulose paper, and radioactivity counting were performed as described (5). The nomenclature proposed by Shaw et al (20) for the aminoglycoside resistance genes and the enzymes they encode is used in this paper. Determination of minimum inhibitory concentration (MIC). MICs were determined by the agar dilution method (1). Four single colonies from each clone were transferred to LB broth and incubated overnight at 37°C with shaking. A 3 pl aliquot of each of the bacterial suspensions (107 cfu/ml; 100x dilution of the overnight culture) was spotted onto two sets of LB agar plates containing 0, 10, 25, 50, 100, 200, or 500 pg streptomycin ml'l. One set of plates was amended with 0.1 mM IPTG. Bacterial strains without confluent growth in the spots after 16 h incubation at 37°C were considered sensitive to the respective level of streptomycin. 50 Deletion analysis in the strA and strB region. A 2.8-kb XbaI-PstI fragment from pEa34, with the strA-strB genes and a promoter derived from I81133, was cloned into the XbaI- PstI site of pGEM3Zf(+), downstream from a lac promoter, to produce plasmid pSTRXP. Deletions in the strA and strB region were performed with the Erase-a-Base System (Promega Corp., Madison, WI). The deletion derivatives were transformed into competent E. coli JM109 cells (15). The extent of each deletion was verified by DNA sequencing. MICs and in vitro APH enzyme activity for each clone were determined as described above. In the complementation assay, deletion derivative pB39 was transformed into E. coli JM109 harboring pC15 or pE21. Transformants were selected on LB agar amended with 100 pg streptomycin ml’l. Four transformed clones from the two complementation assays were selected and their MICs determined as described above. Modification of strB and cloning into pTWNHE. A series of primers were used to modify and amplify strB by the polymerase chain reaction (PCR; Table 1). The forwarded primers AJ24 to AJ28, and primer AJ34 contained a potential Shine-Dalgarno sequence with 6 to 9 nucleotides complementary to the 3'-f1anking sequence of the 16S rRNA. The reverse primer AJ23 contained an EcoRI site to facilitate cloning of the amplified DNA. The PCR was run through 30 cycles at 90°C for 1 min, 50°C for 1 min, and 72° C for 1 min. The reaction mixture (100 p1) consisted of 200 pmol each of two primers (AJ23 and one of AJ24, AJ25, AJ26, 51 AJ27, AJ28, and AJ34), 0.01 ng of plasmid pC15 as template, 10 p1 of 10X Taq reaction buffer (200 mM Tris.HCl, pH 8.4, 500 mM KCl), 2.5 mM MgC12, 200 pM of each of four dNTPs, and 2.5 units Taq DNA polymerase. The reactions produced 0.84- kb strB variants strB—AJ24, strB-AJZS, strB-AJ26, strB-AJ27, strB-AJ28, and strB-AJ34. They were separated from the reaction mixtures with a DNA purification system kit (Magic PCR Preps, Promega Corp., Madison, WI), digested with EcoRI, and then cloned into the HindIII-EcoRI site of pTWNHE, 46-bp downstream from the tac promoter. Modifications in each strB variant were examined by DNA sequencing with primer AJ30, which complemented the sequence 28-bp upstream from the HindIII site of pTWNHE. Variant strB-AJ26N was made by filling-in the NotI site of strB—AJ26. It encoded a polypeptide with 14 additional amino acids than the authentic strB protein due to a frameshift at codon 268. Variants strA-B39 and strB-C15 were HindIII-EcoRI fragments from pB39 and pC15 with wild-type strA and strB coding sequences, respectively. They were cloned into the HindIII- EcoRI site of pTWNHE for expression analysis. Expression and analysis of strA and strB gene products. Each clone was grown in LB broth at 37°C with shaking until the optical density at 600 nm was 0.6 and then IPTG was added (final concentration 0.5 mM). No IPTG was added to the controls. The bacteria were incubated for an additional 4 h and harvested by centrifugation. The cell pellets were resuspended in 1/5 volumes of sample buffer (62.5 mM 52 Tris.HCl pH 6.8, 2% sodium dodecyl sulfate [SDS], 10% glycerol, 5% B-mercaptoethanol, and 0.002% bromophenol blue), heated for 10 min at 90°C, and the proteins separated by electrophoresis on 13.5% SDS-polyacrylamide gels (12). N-terminal amino acid sequence determination. The protein encoded by strA from strain SMAB39 and those encoded by the strB variants from strains SMBlO and SMBD3 were separated on a 1.5-mm thick 13.5% SDS-polyacrylamide gel. Proteins were then electroblotted onto a polyvinylidene difluoride membrane (14). The proteins were excised and their N- terminal amino acid sequences determined using the automatic Edman degradation method and a 477A Protein Sequencer (Applied Biosystems, Foster City, CA) at the Macromolecular Facility Laboratory, Michigan State University. Preparation and 13c-NMR analysis of the phosphorylated streptomycin. E. coli SMA839 and SMBlO were grown in 500 ml of LB broth with IPTG until the optical density at 600 nm was 1.0. Crude enzyme extracts were prepared by ultrasonication (5). To prepare phosphorylated streptomycin, 50 m1 of each crude enzyme extract was added to an end volume of 150 ml of a mixture of 10 mM streptomycin sulfate, 16.6 mM ATP, 60 mM Tris.HC1, 4 mM M9804, 10 mM B-mercaptoethanol, and 0.25 mM phenyl-methyl- sulfonyl-fluoride (PMSF), pH 8. After the reaction mixture was incubated for 48 h at 30°C, protein was removed by ultra-filtration (Diaflo YM3, Amicon Corp., Beverly, MD) and the streptomycin phosphate purified over a 2.5 x 30 cm 53 Amberlite C650 column eluted with a 2N KCl gradient. Fractions containing streptomycin phosphate were detected using the maltol assay (18) and then pooled. The excess KCl salt was precipitated with 3.5 volumes of acetone at pH 2 (24). After removal of the precipitated KCl salt, the filtrate was titrated with 1N KOH until a streptomycin phosphate precipitate formed. The precipitate was collected, dissolved in 10 ml H20, adjusted with 1N HCl to pH 6, and lyophilized. 13C-NMR studies of the respective streptomycin phosphate (The Max T. Rogers NMR Facility, VXR 500 spectrometer; Michigan State University) were conducted in D20 with dioxane as the external standard. Secondary structures. The stem—loop structures and free energy for strA and strB were determined using the computer program Hairpin Loop Search, DNASIS Version 3.0 (Hitachi Software Engineering Co. LTD). RESULTS Deletion and complementation analysis of strA and strB. Plasmids pSTRXP and pC2 contained strA-strB and a promoter from I31133, while plasmid pF16 contained strA-strB but no 181133 promoter (Fig. 1A). Cells of E. coli JM109 transformed with pSTRXP and pC2 were as resistant to streptomycin under both inducing and noninducing conditions as cells transformed with E. amylovora plasmid pEa34. Cells with pF16 were less resistant (MIC = 100 pg streptomycin ml‘ 1) under noninducing than under inducing (MIC >500 pg streptomycin ml'l) conditions. Under inducing conditions, APH enzyme activity was high in extracts from cells with pSTRXP, pC2, and pF16, but low in extracts from cells with the low-copy-number plasmid pEa34. Cells transformed with pC8 and pC15, plasmids with deletions from the 5' end of the strA region, exhibited a dramatic loss of resistance and APH enzyme activity. Cells transformed with pE21, which had a 272-bp NruI fragment deleted from strA, and cells transformed with pA17, a plasmid containing deletions in both strA and strB, were as susceptible to streptomycin as cells transformed with pGEM3Zf(+). Phosphorylation activity in extracts from these cells was low. Cells with p85 and pB39, plasmids with strA and the I81133 promoter but 54 55 Fig. 1. A. Resistance to streptomycin and aminoglycoside phosphotransferase (APH) activity in extracts from cells of Escherichia coli JM109 transformed with various deletions in the strA and strB genes from Erwinia amylovora strain CA11. Deletion derivatives were cloned in vector pGEMBZf(+) with the genes oriented downstream from a lac promoter. Bacteria were grown in the presence and absence of isopropyl-B-D- thiogalactopyranoside (IPTG). Bold lines with arrows represent the coding regions of strA and strB and the transcriptional direction for each gene. Numbers above the lines are nucleotide sequence numbers for Tn5393 (4) and "P" indicates a promoter from I81133. B. Complementation of deletions in strA and strB. A. Deletion study Plasmid pSTRXP pC2 pF16 pE21 pC8 pCl S pA17 p85 p839 pEa34 pGEM3Zf(+) '56 B. Complementation study pB39 + pC15 pB39 + pE21 MIC APH (pg streptomycin (relative ml. activity) 0.5 mM IPTG 4827 4960 5763 5763 - + 83\: / SM N SW :9;— > 500 > 500 52.3 i 3.0 4923 ) )— > 500 > 500 70.0 i 1.0 ' )— > 500 100 70.1 i 2.1 5063 5338 pg )— <10 <10 24:04 4997 >—-— 25 < 10 4.0 i- 0.3 54") 6115 >—— 25 <10 3.8i1.3 L—-)— <10 <10 1.1102 6563 p ) J 100 100 80.5 i 0.5 p )---9‘90 100 100 80.0 i 3.2 p ) )—— > 500 > 500 4.4 :l: 0.1 < 10 < 10 1.0 i- 0.1 500 500 ND 500 500 ND 57 nucleotide deletions in strB, were S-fold less resistant than cells containing pSTRXP or pC2 under both inducing and noninducing conditions. But, extracts from these cells exhibited a high level of APH enzyme activity. When cells were transformed with two plasmids, one with functional strB (pE21 or pC15) and the other with functional strA (pB39), resistance was restored to MIC = 500 pg streptomycin ml"1 (Fig. 18). Effects of Shine-Dalgarno sequence and mRNA secondary structure on the expression of strB. When induced with IPTG, strain SMA839(pTWNHE::strA-B39) produced the expected 27-kDa encoded polypeptide in abundance (13% of the total cellular protein), but strain SMBC15(pTWNHE::strB—C15) did not produce the expected 28-kDa encoded polypeptide in abundance (Fig. 2, lanes strA-B39 and strB-C15). Analysis of the primary sequences for strA and strB revealed that strA contained a Shine-Dalgarno consensus sequence of AAGGA in the ribosome-binding site while strB lacked a Shine- Dalgarno consensus sequence (Fig. 3). Introducing TAAGGA into strB 7-bp upstream from the initiation codon did not increase the amount of the 28-kDa protein produced by strain SMBEl nor affect its resistance to streptomycin (Fig. 2, lane strB-AJ24). When the computer-predicted mRNA secondary structures for strA and strB were assessed, stable stem-loop structures surrounded the ribosome-binding site (Fig. 3, strA-839 and strB-015). The Shine-Dalgarno sequence and the initiation codon for strA were located in the single- 58 Fig. 2. A. SDS-PAGE analysis of proteins from strains of Escherichia coli transformed with expression vector pTWNHE with strA, strB, or modified strB variants. Proteins were separated on a 13.5% SDS-polyacrylamide gels and stained with Coomassie blue. Bands labeled 27, 28, and 30 kDa are protein products from strA, strB, and strB-AJ26N, respectively. B. The percentage of the expressed proteins from strA and strB variants to the total cellular protein for strains grown in LB medium with (+) or without (-) IPTG. Data were obtained by scanning four individual lanes for each strain on SDS gels using AMBIS Core Software Version 4.0 (AMBIS, Inc. San Diego, California). C. Resistance of each strain to streptomycin on LB agar with (+) and without (-) IPTG. A. E. coli strains and gene variants 8. Expression by E. coli strains (% of total cellular protein) c. MIC (pg _ streptomycin ml ) 59 strA- strB- strB- strB- strB- strB- strB- strB- strB- 839 C15 AJ24 AJ34 AJ25 AJ26 AJ26N AJ27 AJ28 SMABSQ SMBC15 SMBE‘I SMBCS SMB1 SMBlO SMBlON SMBAQ SMBDS 60 Fig. 3. Predicted mRNA secondary structures surrounding the ribosome-binding site for strA, strB, and six strB variants. The stem-loop structures and free energy (AG; in kcal mol' 1) were determined with the computer program Hairpin Loop Search, DNASIS version 3.00 (Hitachi Software Engineering Co. LTD). Nucleotides in ribosome-binding sites that can pair with the 3' end of E. coli 16S rRNA (AUUCCUCCACU...5') are indicated as larger characters and those that cannot pair with strB sequence are underlined. Initiation codons are enclosed by a box and the stop codon from strA is marked with asterisks. The secondary structures for strB-AJ24 and strB-AJ34 were unaltered, the structures for strB-AJZS, strB-AJ26, strB-AJ27, and strB-AJ28 were altered by replacing nucleotides between the Shine-Dalgarno sequence and the initiation codon. Six nucleotides (the first two codons of strB) on strB-AJ27 and strB-AJZB were omitted. The strain of Escherichia coli transformed with each gene is shown in parenthesis under the gene designation. strA-B39 a-u A-U a-u (SMABB9 ) c u A-U cc 0 c A-U c-c U-A u-a c 0 ese G=C C 5 ‘ -GGUUGAGCCACCUC A UUCUGACUGGU-3 ' U U as U G U OGO C A CG 00 strB-M24 c-c G ( SMBE 1 ) g-A 5 'CUCUAAGGAAG 0 AG = -15.94 on Us 0 c c , u U c c C u A strB-M25 GC_GU (SMBI) 3:3 .‘Hc 5 - UMGGAGUUAACGUUIAUG Iooc cunncecnccoc—a' UC U0 CU G U G U 0:0 C A C-G c-c Eli 5 ° UMGGAGUUAACGUU ccwocccAAccuc-a' AG - -5.74 strB-M2 7 (SMBA9 ) GUUCUCAUUGCGGA-3 ' 61 U U Us 0 c c u U c cCG Una c-c strB-C15 as c (SMBC 15 ) EA c c-c u u u u U c G * we a U-A U-A G=C c-c 5 ' —ccAcccucucacuch u GuucucauuccccA—a ' U U U C CU G U C c u u c CCG 00A strB-AJ 34 c-c C=G (SMBCS ) G=C U-A A c c=c u u u u u c c c-c U-A U-A sac GGgCU 5 ' UAAGGAGGUA GUUCUCAUUGCGGA-3' c U c c u u c c A c-G (SMB1 O ) c-c c-c U-A 5 - UAAGGAGGUUMCGUU [flue 00c“ CGUUUCGCMCCUG-a' G U c c u U c C A strB-11.728 C c C_Gu U (SMBD3) [if A C 5 ' UMGGAGGUUAACGUU GUUUCGCAACCUG-3 ' 62 stranded region but the initiation codon for strB was located in the double-stranded region of the stem-loop structure. To assess the effect of the strength of the Shine-Dalgarno sequence on the expression of strB, the sequences TAAGGAG (primers AJ25 and AJ27) and TAAGGAGGT (primers AJ26, AJ28, and AJ34) were introduced into the sequence 7- and 8-bp upstream from the initiation codon. To study the effect of the stem-loop structure on expression of strB, the structure was altered by nucleotide replacement. Each of the stem- loop structures of strB-AJ25, strB-AJ26, strB-AJ27, and strB-AJ28 had a free energy less negative (AG = -5.74 kcal mol‘l, Fig. 3) than that for strB-AJ34 (AG = —15.94 kcal mol'l). The initiation codons for strB-AJ25 and strB-AJ26 were free from the stem-loop, those for strB-AJ27 and strB- AJ28 were located, due to the removal of two codons ATGTTC, in the double-stranded region of the stem-loop structure. When induced with IPTG, strains SMBl, SMBlO, SMBD3, and SMBC5 were resistant to 100 pg streptomycin ml‘1 and produced the 28-kDa polypeptide at levels of 12, 16, 4, and 4% of total cellular protein, respectively (Fig. 2, lanes strB-AJ25, strB-AJ26, strB-AJ27, and strB-AJ28). Strains SMBlO, SMBD3, and SMBC5 with Shine-Dalgarno sequence TAAGGAGGT produced more strB protein than strains SMBl and SMBA9 with Shine-Dalgarno sequence TAAGGAG. Strain SMBl (initiation codon free from the stem) produced more gene product than strains SMBC5 and SMBDB (initiation 63 codon located in the stem) even though strains SMBC5 and SMBD3 contained a longer Shine-Dalgarno sequence. Overproduction of strB protein in strains SMBl, SMBlO, SMBD3, and SMBC5 was accompanied with an increase in the level of resistance, but MICs did not increase above 100 pg streptomycin ml'1 even though the encoded polypeptide increased from 4 to 16% of the total protein. Strain SMBlON produced a 30-kDa protein (with a modification in the C- terminal of strB protein) consisting of 16% of the total protein, but was sensitive to streptomycin (Fig. 2. lane strB-AJZ 6N) . Confirmation of strA and strB proteins. The first 12 N- terminal amino acids of the protein from E. coli SMAB39 were consistent with the amino acid sequence predicted from the nucleotide sequence for strA (4, 19). The first 11 N- terminal amino acids of the proteins from E. coli SMBlO and SMBD3 were M-F-M-P-P-V-F-P-A-H-W-H and M-P-P-V-F-P-A-H-W—H- V-S. These sequences were identical to the amino acid sequences predicted from the nucleotide sequences for strB- AJ26 and strB-AJ28, respectively. Identification of the phosphorylated streptomycin products. The 13C-NMR spectrum for the 21 carbons on the streptomycin phosphate produced by incubating streptomycin with strA encoded protein was consistent with the spectrum for streptomycin-3"-phosphate (8, 11). The difference in chemical shift for carbon position C3" of streptomycin and of phosphorylated streptomycin was 2.8 ppm and the signals 64 at carbons C2", C3", and C4" were split by coupling with the phosphorous group (coupling constants were 2.6 Hz at C2", 5.3 Hz at C3", and 3.1 Hz at C4"). Therefore, the enzyme encoded by strA was APH(3")-Ib. The 13C-NMR spectrum for the streptomycin phosphate produced when streptomycin was reacted with strB encoded protein showed split chemical shifts at carbons C6 and C1 (coupling constants were 5.3 Hz at C6 and 3.2 Hz at C1). The difference of 3.5 ppm in the chemical shifts at carbon C6 between streptomycin and the phosphorylated streptomycin was similar to that reported for carbon C6 in streptomycin-G-phosphate (23). This established that the enzyme encoded by strB was APH(6)-Id. DISCUSSION Our biochemical analysis of the enzymes produced by strA and strB support predictions made from protein sequence data (20) that the strA-strB genes encode two aminoglycoside phosphotransferases, APH(3")-lb and APH(6)-Id, respectively. We also confirmed in the deletion and complementation studies that both genes were required for a high level of resistance to streptomycin. Interaction between APH(3")-1b and APH(6)-Id appear essential for high resistance since bacteria with only strA or strB were 2.5 to 5 fold more sensitive to streptomycin than bacteria with both genes. When strA and strB were cloned separately, only strA was expressed. Examination of the primary sequence data revealed that strA-strB were overlapping genes and that the genes were translationally coupled. When strB was separated from strA by cloning, cloned strB was devoid of an reinitiation region and translation was inhibited until a suitable Shine-Dalgarno sequence was inserted 5' of the initiation codon. It is also possible that the differential expression of strA-strB is regulated by mRNA secondary structure surrounding the ribosome binding site. The predicted stem- loop structure for strA mRNA revealed that the Shine- 65 66 Dalgarno sequence and the initiation codon, two elements within the ribosome-binding site that allow translation initiation (9), were located in the single-stranded region of a stable stem-loop structure. However, no Shine-Dalgarno sequence was present in the predicted stem-loop structure for strB and the initiation codon was located in the double- stranded region of a stable stem-loop structure. We found that a weak Shine-Dalgarno sequence in the single-stranded region upstream from an initiation codon in the double- stranded region was not sufficient for APH(6)-Id production. When the Shine-Dalgarno sequence and the initiation codon were both located in the single-stranded region, APH(6)-Id production was maximized and bacteria with this DNA were resistant to streptomycin. The gene encoding APH(6)-Ia, but not the one encoding APH(3")-Ia, is part of the gene cluster involved in streptomycin biosynthesis in the streptomycin-producing Streptomyces griseus (6, 11). It was suggested that APH(6)- Ia may protect S. griseus from the toxic action of streptomycin (17, 22). However, the biological function of APH(3")-Ia in S. griseus is unclear. Our study suggests that the coexistence of APH(3")-Ia and APH(6)-Ia in S. griseus may enable it to tolerate high levels of streptomycin just as the coexistence of APH(3")-Ib and APH(6)-Id in bacteria enable them to tolerate high levels of streptomycin. ACKNOWLEDGMENTS This research was supported by the Michigan Agricultural Experiment Station and the Michigan Apple Research Committee. The NMR data were obtained on instrumentation that was purchased in part with funds from NIH grant #1-810- RR04750, NSF grant fCHE-88-00770, and NSF grant #CHE-QZ- 13241. 67 REFERENCES 1. Barry, A. L. 1976. The antimicrobic susceptibility test: principles and practices. The Lea & Febiger, Philadelphia. 2. Burr, T. J., J. L. Norelli, C. L. Reid, L. K. Capron, L. 8. Nelson, H. 8. Aldwinckle, and N. F. Wilcox. 1993. Streptomycin-resistant bacteria associated with fire blight infections. Plant Dis. 77:63-66. 3. Chiou, C.-8., and A. L. Jones. 1991. The analysis of plasmid-mediated streptomycin resistance in Erwinia amylovora. Phytopathology 81:710-714. 4. Chiou, C.-8., and A. L. Jones. 1993. Nucleotide sequence analysis of a transposon (Tn5393) carrying streptomycin resistance genes in Erwinia amylovora and other gram- negative bacteria. J. Bacteriol. 175:732-740. 5. Davies, J. E. 1986. Aminoglycoside-aminocyclitol antibiotics and their modifying enzymes. p. 790-809. In V. Lorian (ed.). Antibiotics in Laboratory Medicine. The Williams & Wilkins Co., Baltimore. 6. Distler, J., A. Ebert, K. Mansouri, R. Pissowotzki, M. stockmann, and w. Piepersberg. 1987. Gene cluster for streptomycin biosynthesis in Streptomyces griseus: nucleotide sequence of three genes and analysis of transcriptional activity. Nucl. Acids Res. 15:8041-8056. 7. Furste, J. P., w. Pansegrau, R. Frank, H. Blocker, P. 8cholz, M. Bagdasarian, and E. Lanka. 1986. Molecular cloning of the plasmid RP4 primase region in a multi-host- range tacP expression vector. Gene 48:119-131. 8. Ganelin v. L., L. G. Vinogradova, V. N. stepanshina, R. M., Pstyushenko, A. I., Chernyshev, 8. E. Esipov, Y. o. Bazykin, and 8. M. Navashin. 1980. Streptomycin-3"- phosphotransferases from streptomycin-resistant cells of Escherichia coli strains. Biochimia 45:2198-2205. 9. Gold, L. 1988. Posttranscriptional regulatory mechanisms in EScherichia coli. Ann. Rev. Biochem. 57:199-233. 10. Guarry, P., J. van Embden, and 8. Falkow. 1974. Molecular nature of two nonconjugative plasmids carrying 68 69 drug resistance genes. J. Bacteriol. 117:619-630. 11. Heinzel, P., O. Herbitzky, J. Distler, and W. Piepersberg. 1988. A second streptomycin resistance gene from Streptomyces griseus codes for streptomycin-3"- phosphotransferase. Arch. Microbiol. 150:184-192. 12. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685. 13. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 14. Matsudaira, P. 1987. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J. Biol. Chem. 262:10035-10038. 15. Nishimura, A., M. Morita, Y. Nishimura, and Y. Engine. 1990. A rapid and highly efficient method for preparation of competent Escherichia coli cells. Nucleic Acids Res. 1836169. 16. Norelli, J. L., T. J. Burr, A. M. Lo Cicero, M. T. Gilbert, and B. H. Katz. 1991. Homologous streptomycin resistance gene present among diverse gram-negative bacteria in New York State apple orchards. Appl. Environ. Microbiol. 57:486-491. 17. Piwowarski, J. M., and P. D. Shaw. 1979. Streptomycin resistance in a streptomycin-producing microorganism. Antimicrob. Agents Chemother. 16:176-182. 18. Schenk, J. R., and M. A. Spielman. 1945. The formation of maltol by the degradation of streptomycin. J. Am. Chem. Soc. 67:2276-2277. 19. 8cholz, P., V. Haring, B. Wittmann-Liebold, R. Ashman, M. Bagdasarian, and E. 8cherzinger. 1989. Complete nucleotide sequence and gene organization of the broad-host- range plasmid RSF1010. Gene 75:271-288. 20. Shaw, x. J., F. N. Rather, R. 8. Hare, and G. H. Miller. 1993. Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes. Microbiol. Reviews 57:138-163. 21. aobiczewski, P., C.-8. Chiou, and A. L. Jones. 1991. Streptomycin-resistant epiphytic bacteria with homologous DNA for streptomycin resistance in Michigan apple orchards. Plant Dis. 75:1110-1113. 22. Sugiyama, M., H. Mochizuki, 0. Mini, and R. Nomi. 1981. 7O Mechanism of protection of protein synthesis against streptomycin inhibition in a producing strain. J. Antibiot. 34:1183-1188. 23. Tohyama, B., T. Shigyo, and Y. Okami. 1984. Cloning of streptomycin resistance gene from a streptomycin producing streptomycete. J. Antibiot. 37:1736-1737. 24. vander Brook, M. J., A. N. Wick, w. H. DeVries, R. Harris, and G. F. Cartland. 1946. Extraction and purification of streptomycin, with a note on streptothricin. J. Biol. Chem. 165:463-468. 25. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103-119. APPENDIX A PROPOSED RESEARCH ON RIBOSOME-HEDIATED STREPTOMYCIN RESISTANCE IN Erwinia amylovora PROPOSED RESEARCH ON RIBOSOME-MEDIATED STREPTOMYCIN RESISTANCE IN Erwinia amylovora INTRODUCTION Fire blight, caused by Erwinia amylovora, is the most important and destructive bacterial disease of apples and pears in North America, New Zealand, much of Europe, and the Mediterranean region (29). The methods for control of fire blight are difficult, expensive, and not always effective. Streptomycin is the most reliable and effective bactericide for the control of fire blight. However, intensive use of streptomycin has been accompanied by the development of resistant strains of E. amylovora (18, 2, 13, 26). Streptomycin-resistant E. amylovora were first detected in Michigan in an apple orchard in Van Buren county in 1990 (Part I, this thesis). By 1993, resistant strains had been detected in eight more apple orchards in Van Buren county, two orchards in Kent county, and three orchards in Newaygo county (14). Bacteria use three strategies to cope with streptomycin: alteration of the ribosomal target site, inactivation of streptomycin by modifying enzymes, and reduced accumulation of streptomycin in the cells. The enzyme- and ribosome- mediated mechanisms are considered to be the most important 72 73 of the three mechanisms because they are associated with high levels of resistance. Among the streptomycin-resistant strains of E. amylovora isolated from Michigan apple orchards some exhibit an aminoglycoside phosphotransferase (APH) activity in enzyme assays, while other strains do not exhibit the enzyme activity. The molecular genetics and biochemistry of resistance in strains with the APH enzyme was the subject of this thesis. The resistant strains lacking APH activity exhibit cross resistance to myomycin, an antibiotic with a mode of action similar to streptomycin. Strains resistant to both streptomycin and myomycin often have mutations in ribosomal genes (3). Although enzyme- mediated resistance is more common than ribosome-mediated resistance in E. amylovora from Michigan, ribosome-mediated resistance is common in Washington, Oregon (13), and California (25). I propose to determine whether resistance in these strains of E. amylovora is due to mutations in ribosomal components, whether all field strains have the same or different mutations, and whether strains with different mutation sites have different levels of resistance and protein synthesis. I also propose to determine the nucleotide sequence for the 16S rRNA and r-protein S12 genes of E. amylovora. OBJECTIVES 1. To test whether there are different resistance levels 74 present in the myomycin- and streptomycin-resistant strains of E. amylovora. 2. To determine the molecular basis and biochemical mechanism for streptomycin reSistance in these strains. 3. To determine whether resistant field strains with altered ribosomes have lower levels of protein synthesis in vitro and lower growth rates than streptomycin—sensitive strains. JUSTIFICATION Developing an understanding of the mechanism of streptomycin resistance in E. amylovora may suggest methods for combating resistant strains. In this research project, the resistance mechanism will be investigated by comparing the nucleotide sequences of 16S rRNA and r-protein 812 genes from sensitive and the myomycin- and streptomycin-resistant E. amylovora and by determining the sensitivity of ribosomes from these strains to streptomycin in an in vitro protein synthesis assay. Although mutations in many ribosomal components and sites may result in insensitivity to streptomycin, mutations in the field strains may be limited to one or a few sites. If there is only one mutation site, the information suggests that the most stable and fittest mutation is unique. Thus, we can design specific primers for the detection of the point mutation by PCR-based methods. If mutation occurs in several sites on the ribosome that can be reflective to distinct resistance levels in the bacterial cells. The alteration on ribosome 75 may impair the protein synthesis and competition of the niche of the cell. This information is useful for the prediction for fitness of the resistant strains in nature. Although mutations for streptomycin resistance may occur in several sites in r-protein S4, 85 and $12, and 16S rRNA, mutations in r-protein $12 and 16S rRNA are the most common. Resistance due to mutations on r-protein $12 and 16S rRNA has been reported not only in bacteria (5, 27) but also in chloroplast of plants and green blue algae (6, 7, 12, 20). Most mutations in the 16S rRNA gene are in the domains 530- loop and 915 region (Escherichia coli numbering) (5, 11, 16, 19). The different levels of streptomycin resistance may result from mutations at different positions of r-protein 812 or 16S rRNA. Mutations at different positions of $12 and 16S rRNA genes having different levels of resistance have been reported (22, 24). If the field strains of E. amylovora exhibit different levels of resistance to streptomycin, they may have different mutation sites in r-protein S12 or 16S rRNA. Ribosomes with mutated r-protein 812 gene often exhibit impaired protein synthesis in vitro. The efficiency of a natural messenger RNA (M82 RNA)-directed protein synthesis is about 50% of the wild type (11). The impaired ribosome may slow the growth of the bacterium and be harmful to its fitness. 76 In a preliminary study, the streptomycin-resistant strains of E. amylovora which did not exhibit APH activity were found to be resistant to the antibiotic myomycin, while strains with APH activity were sensitive to myomycin. Except for plasmid pEA29, the myomycin- and streptomycin- resistant strains tested contain no other plasmid. Resistance in these strains may be chromosomal. It may result from mutations in the genes of ribosomal components S12 and 16S rRNA. DESCRIPTION OF RESEARCH PLAN To test if there are various resistance levels present in myomycin- and streptomycin-resistant strains of E. amylovora isolated from Michigan and other States, the minimum inhibitory concentration (MIC) will be determined in King's medium B (KB) (10) amended with 0.1, 0.5, 1, 2, 4, 6, 8, and 10 mg streptomycin ml'l. If different resistance levels are present, two or three strains from each level will be selected for further analysis. Otherwise, 30 strains will be selected from the pool of resistant E. amylovora. To clone the $12 gene a 0.6-kb fragment containing the complete S12 gene will be amplified from the selected resistant strains and a sensitive strain EL01 and a APH- producing resistant strain CA11 of E. amylovora using the polymerase chain reaction (PCR). Forward primer GGCCTGGTGATGATGGCGGG in the 5' noncoding region of 812 gene and the reverse primer CGTGGCATGGAAATACTCCG which 77 complementary to the neighboring S7 gene, will be synthesized for the PCR amplification. The two primers were designed based on the consensus sequences in the two cloned 812-S7 genes from E. coli and Salmonella typhimurium (9, 23), which with E. amylovora belong to the family Ehterobacteriaceae. The genomic DNA from the E. amylovora strains will be prepared (28) and the PCR reactions will be conducted as the described in the Part III of this thesis. To clone the 16S rRNA gene a 0.6-kb DNA fragment will be amplified by PCR and used as probe. Forward primer GCACAATGGGCGCAAGCCTG and reverse primer GGTAAGGTTCTTCGCGTTGC will be used in the PCR amplification. These primers were designed based on the consensus sequences of 168 rRNA genes from E. coli, Yersinia enterocolitica, and Erwinia carotovora (1, GenBank Accession number M59149 and M59292). Genomic DNA from E. amylovora EL01 will be prepared and a cosmid genomic library will be constructed in vector pHC79 (8). Colony hybridization with the probe will be conducted to identify and isolate recombinant clones containing the 16S rRNA gene. The complete nucleotide sequence of 168 rRNA gene from E. amylovora EL01 will be determined and used as the standard for sequence comparison. Since the 0.6-kb fragment contains the 530-loop and 915 region, the two primers can be used not only for DNA amplification from the resistant strains and strains CA11 but also for direct sequencing of the amplified fragments. The sequences of r- protein 812 and 16S rRNA from strain EL01 and the resistant 78 strains will be compared using the FASTA program of the Genetics Computer Group Sequence Analysis Software Package, Version 7 (4). If the mutation site in the resistant strains is unique, a specific primer will be synthesized based on the principle of the amplification refractory mutation system (ARMS; 21) for detection of the point mutation by PCR. The efficiency of protein synthesis and the error frequency between normal and altered ribosomes will be assayed. Ribosomes will be isolated by sucrose-gradient centrifugation (17) from sensitive strain EL01 and resistant strains of E. amylovora. Protein synthesis efficiency will be tested under the direction of a natural messenger, M82 RNA in the absence or the presence of streptomycin (17). To establish error frequency, I will measure the ratio of isoleucine incorporated per phenylalanine under the direction of poly(U) in the presence of streptomycin (17). To test whether E. amylovora with altered ribosomes is incompetent to the cell with wild-type ribosome, competition between a mutant and a wild-type E. amylovora in rich medium and minimum medium will be conducted. The growth curve of strain EL01 and mutants will be determined in KB medium and in minimum medium M9 (15). To test the competition between the wild type and ribosome-altered mutants, EL01 and one of the tested mutants will be co-cultivated in the KB medium and the M9 medium and the growth curve of both strains will be determined. 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Bottger. 1993. Molecular basis of streptomycin resistance in Mycobacterium tuberculosis: alterations of the ribosomal protein 812 gene and point mutations within a functional 168 ribosomal RNA pseudoknot. Mol. Microbiol. 9:1239-1246. 6. Galili, 8., H. Fromm, D. Aviv, M. Edelman, and E. Galun. 1989. Ribosomal protein 812 as a site for streptomycin resistance in Nicotiana chloroplasts. Mol. Gen. Genet. 218:289-292. 7. Harris, E. E., B. D. Burkhart, N. I. Gillham, and J. E. Boynton. 1989. Antibiotic resistance mutations in the chloroplast 168 and 238 rRNA genes of Chlamydomonas reinhardii: correlation of genetic and physical maps of the chloroplast genome. Genetics 123:281-292. 8. Bohn, B., and J. Collins. 1980. A small cosmid for efficient cloning of large DNA fragments. Gene 11:291-298. 9. Hughes, D., and R. B. Buckingham. 1991. The nucleotide sequence of rpsL and its flanking region in Salmonella typhimurium. Gene 104:123-124. 80 81 10. King, E. 0., M. R. Ward, and D. E. Raney. 1954. Two simple media for the demonstration of pyocyanin and fluorescin. J. Lab. Clin. Med. 44:301- -307. 11. Leclerc, D., P. Melancon, and L. Brakier-Gingras. 1991. Mutations in the 915 region of Escherichia coli 168 ribosomal RNA reduce the binding of streptomycin to the ribosome. Nucl. Acids Res. 19:3973-3977. 12. Liu, x.-Q., N. I. Gillham, and J. E. Boynton. 1989. Chloroplast ribosomal protein gene rpslz of Chlamydomonas reinhardtii: wild- -type sequence, mutation to streptomycin resistance and dependence, and function in Escherichia coli. 264:16100- -16108. 13. Loper, J. E., M. D. Eenkels, R. G. Roberts, G. G. Grove, M. J. Willett, and T. J. Smith. 1991. Evaluation of streptomycin, oxytetracycline, and copper resistance of Erwinia amylovora isolated from pear orchards in Washington State. Plant Dis. 75:287- 290. 14. McManus, P., and A. L. Jones. 1993. Epidemiology and genetic analysis of streptomycin-resistant Erwinia amylovora from Michigan apple orchards and evaluation of oxytetracycline for control. (Submitted) 15. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 16. Melancon, P., C. Lemieux, and L. Brakier-Gingras. 1988. A mutation in the 530 100p of Escherichia coli 168 ribosomal RNA causes resistance to streptomycin. Nucl. Acids Res. 16:9631-9639. 17. Melancon, P., D. Leclerc, L. Brakier-Gingras. 1990. A deletion mutation at the 5' end of Escherichia coli 168 ribosomal RNA. Biochim. Biophys. Acta 1050:98-103. 18. Miller, T. D., and M. N. Schroth. 1972. Monitoring the epiphytic population of Erwinia amylovora on pear with a selective medium. Phytopathology. 62:1175-1182. 19. Montandon, P.-E., R. Wagner, and E. Stutz. 1986. E. coli ribosomes with a C912 to U base change in the 16S rRNA are streptomycin resistant. EMBO J. 5:3705-3708. 20. Montandon, P.-E., P. Nicolas, P. Schurmann, and E. Stutz. 1985. Streptomycin-resistance of Euglena gracilis chloroplasts: identification of a point mutation in the 16S rRNA gene in an invariant position. Nucl. Acids Res. 13: 4299- -4310. 82 21. Newton C. R., A. Graham, L. E. Heptinstall, et al. 1989. Analysis of any point mutation in DNA: the amplification refractory mutation system (ARMS). Nucleic Acids Res. 17:2503-16. 22. O'Connor, M., E. A De Stasio, and A. E. Dahlberg. 1991. Interaction between 16S ribosomal RNA and ribosomal protein 812: Differential effects of paromomycin and streptomycin. Biochimie 73:1493-1500. 23. Post, L. E., and M. Nomura. 1980. DNA sequences from the str operon of Escherichia coli. J. Biol. Chem. 255:4660- 4666. 24, Powers, T., and H. F. Noller. 1991. A functional pseudoknot in 168 ribosomal RNA. EMBO J. 10:2203-2214. 25. Schroth, M. M., S. v. Thomson, and w. J. Moller. 1979. Streptomycin resistance in Erwinia amylovora. Phytopathology. 69:565-568. 26. Shaffer, I. B., and R. N. Goodman. 1985. Appearance of streptomycin-resistant Erwinia amylovora in Missouri apple orchards. (Abstr.) Phytopathology. 75:1281. 27. Stuy, J. B., and R. 8. Walter. 1992. Cloning, characterization, and DNA base sequence of the high-level streptomycin resistance gene strAl of Haemophilus influenzae Rd. J. Bacteriol. 174:5604-5608. 28. Wilson, K. 1988. Preparation of genomic DNA from bacteria. Pages 2.4.1-2.4.5. In F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl, (ed.), Current protocols in molecular biology. Vol. 1. John Wiley & Sons, New York. 29. van der Ewet, T., and H. L. Mail. 1979. Fire blight: A bacterial disease of Rosaceous Plants. U. 8. Dep. Agric. Agric. Handb. 510. 200pp. APPENDIX B CONJUGATIONAL TRANSFER OF pEa34 CONJUGATIONAL TRANSFER OF pEa34 Conjugative plasmids are important vehicles for horizontal transfer of new genetic material into plant pathogenic bacteria. Besides the ubiquitous plasmid pEA29 (1), only a 56-kb plasmid has been described in some strains of E. amylovora (2). Plasmid pEa34, the plasmid characterized in this thesis, has not been described previously and appears to be of recent origin in E. amylovora. Therefore, the opportunity for the acquisition of antibiotic resistance via plasmids must be rare in E. amylovora. This may be the reason that plasmid-borne resistance in E. amylovora was not detected until 1990, nearly four decades after streptomycin was registered for the control of fire blight. To be a conjugative plasmid, pEa34 is quite small. Normally, the whole tra gene cluster (encoding proteins for conjugation process) spans about 30 kb. pEa34 must have the whole tra cluster arranged in DNA of less than 28 kb. pEa34 may also be a broad-host-range plasmid. In addition to E. amylovora, pEa34 also exists in E. herbiccla and can move by conjugation from the donors into E. coli and P. syringae pv. papulans (Table 1). The detection of pEa34 in five strains of E. herbicola and the conjugative transfer of the plasmid from E. herbicola 84 85 TABLE 1. The transfer frequency of pEa34 by conjugation among strains of Erwinie amylovore, Escherichia coli, Pseudomones syringes pv. papulans and Erwinie herbicole. Donor Recipient Conjugation Strain Strain Frequency Genetic Marker anda Screening Conditions E. amylovore E. coli CA11 JM109 1.1 DHSa 1.4 JM109(pUCD800) 3.7 JM109(pGEM3Zf(+)) 5.1 E. emylovore P. syringes pv. papulans CA11 Psp32 7 . 9 E. coli E. amylovore JM109(pEa34) scoe 5,0 E. coli E. coli JM109 (pEa34) JM109 (pUCD800) 3 . 2 E. coli P. syringes pv. papulans JM109(pEa34) Psp32 5,3 E. herbicola E. coli 144 JM109(pGEM3Zf(+)) 1.7 E. herbicole E. amylovore 144 secs 1.2 10'2 10‘7 Strr, 37°C Strr, 37°C Strr + Kanr, 37°C Strr + Amp‘, 37°C Strr + Rifr, fluorescent on King's 8 medium Strr + Rifr Strr + Kanr Strr + Rifr, fluorescent on King's B medium Strr + Ampr Strr + Rifr, not 37°C 86 Table 1 (Cont'd) a Strr: streptomycin-resistant; Kent: kanamycin-resistant; Amp‘: ampicillin-resistant; Rift: Rifampicin-resistant; 37°C: screening the transconjugants at 37°C. 87 into E. amylovora suggests the possible movement of pEa34 between the two bacterial species. Dissemination of resistance on conjugative plasmid can be horizontal and vertical, but the dissemination of resistance on chromosome can only be vertical. Therefore, the occurrence of streptomycin resistance on pEa34 in E. amylovora is likely to hasten the spread of resistance into additional Michigan apple orchards via conjugational transfer of pEa34 into sensitive strains of the pathogen. REFERENCES Falkenstein, B., P. Bellemann, 8. Walter, w. Zeller, and x. Geider,. 1988. Identification of Erwinia amylovora, the fireblight pathogen, by colony hybridization with DNA from plasmid pEA29. Appl. Environ. Microbiol. 54:2798-2802. Steinberger, E. M., G.-Y. Cheng, and 8. v. Beer. 1990. Characterization of a 56-kb plasmid of Erwinia amylovora Ea322: its noninvolvement in pathogenicity. Plasmid 24:12- 24. 88 APPENDIX C IDENTIFICATION OF THE MECHANISMS OF STREPTOMYCIN RESISTANCE IDENTIFICATION OF THE MECHANISMS OF STREPTOMYCIN RESISTANCE Among the mechanisms for streptomycin resistance, the resistance mechanisms based on modifying enzymes or on altered ribosomes are more important than the mechanism based on reduced accumulation because the former exhibit high level of resistance. Resistant strains isolated from orchards and that can grow on media plates with 20 pg streptomycin ml'1 or more are considered that resistance in the strains is enzyme-mediated or ribosome-mediated. Streptomycin is inactivated via enzymatic modification by aminoglycoside phosphotransferases, APH(3") and APH(6), and aminoglycoside adenylyltransferases, ANT(3") and ANT(6). The modification is accomplished by transfer of a phosphate group (APH enzymes) or adenyl group (ANT enzymes) from ATP to the 3"- or 6-hydroxyl group (See Fig. 1). myomycin is an broad-spectrum antibiotic produced by a member of the NOcardia genus (3). The mode of action of myomycin in vivo and in vitro closely resembles that of streptomycin. Spontaneous myomycin-resistant mutants of E. coli are essentially indistinguishable from streptomycin- resistant mutants at the ribosomal RNA and ribosomal protein. However, myomycin is not a substrate for the known 90 91 Fig. 1. Molecular structures of streptomycin and spectinomycin and the reaction sites of APH(3"), APH(6), ANT(3"), and ANT(6). 92 Streptomycin NH HN‘C \ NHZ APH( I!) \ Spectinomycin H0 93 streptomycin-modifying enzymes and could be useful in the characterization of natural streptomycin-resistant isolates and in counterselecting against the presence of streptomycin-modifying enzymes. Recent studies on streptomycin resistance have enabled the resistance mechanisms to be characterized in molecular level. Streptomycin resistant isolates can be divided into enzyme- and ribosome-mediated mechanisms by myomycin (Fig. 2). The modifying enzymes, APH and ANT, can be identified by phosphocellulose biding assay using [7-32PJ-ATP and 14C- ATP, respectively, as cosubstrates (1). APH(3") and APH(6) can be distinguished by analyzing the respective phosphorylated streptomycin using 13C-NMR analysis and chemical reactions (Part III, 6). ANT(3") and ANT(6) can be distinguished by the resistance to spectinomycin (2, 4) or analyzing their adenylylated streptomycin with complicated chemical methods (8). However, the easiest way for identifying the enzyme-mediated mechanism is to probe the existence of the Specific genes which have been cloned (7). Since altered ribosome is insensitive to streptomycin in protein synthesis and decoding of translation (5), to confirm the ribosome-mediated mechanism the sensitivity of ribosome to streptomycin in protein synthesis needs to be determined. Although alteration of several ribosomal subunits can reduce the affinity of ribosome to streptomycin, only mutations on 16S rRNA and ribosomal protein 512 are common. To characterize this mechanism 94 Streptomycin-Resistant Isolates Myomycin Resistance? (Yes) (No) Ribosome-Mediated?! Enzyme-Mediated!? Ribosome insensitive APH or ANT to Streptomycin? Activity? (No) (Yes) (Neither) New Mechanism? New Mechanism? * APH(3") ANT(3") Nucleotide Sequence .APE(6) ANT(6) Comparison 13 C-NMR Mutation in Mutation in Spectlnomycln . Resistant? 16$ rRNA Ribosomal APH(3") APH(6) Protein 812 ./’//\\‘K (Yes) (No) ANT(3") ANT(6) Fig. 2. Flowchart for identification of streptomycin resistance mechanisms 95 comparisons of the nucleotide sequences of the ribosome component genes from sensitive and resistant strain and test of the insensitivity of ribosome to streptomycin are required. Studies with myomycin on the bacteria isolated from Michigan and New York State apple orchards have shown that myomycin can counterselect the resistance with modifying enzymes. E. amylovora CA11, P. syringae pv. papulans Psp36, P. asruginosa 60, and E. herbicola 144, which exhibit an aminoglycoside phosphotransferase activity and contain DNA that hybridize to probe SMP3, are sensitive to myomycin (Table 1). The other resistant strains which contain no DNA homologous to the SMP3 are myomycin-resistant. The myomycin-resistant strains may have an altered ribosome because they are also highly resistant to streptomycin. 96 TABLE 1. Characterization of mechanisms of streptomycin resistance by the ability of resistance to streptomycin and myomycin, hybridization to SMP3, and in vitro streptomycin phosphotransferase (APH) and streptomycin adenylyltransferase (ANT) essay. Enzyme Activityb Streptomycin- Myomycin- Hybridization Designation Resistant Resistant to SMP3a APH ANT Erwinie amylovore EL01, BC06 No No No No No CA11, HT62-1 Yes No Yes Yes No R11, 85, Ea88 Yes Yes No No No E. herbicola 6a Yes No Yes Yes No 180 Yes Yes No No No Pseudomones syringae pv. papulans Psp32 No No No No No Psp36 Yes No Yes Yes No MC37, MC38 Yes Yes No No No Pseudomones esruginosa 60 Yes No Yes Yes No a A 0.5-kb tnpR-strA region from P. syringes pv. papulans Psp36. b APH: aminoglycoside phosphotransferase; ANT: aminoglycoside nucleotidyltransferase. REFERENCES 1. Davies, J. 1986. Aminoglycoside-aminocyclitol antibiotics and their modifying enzymes, p. 790-809 In: V. Lorian (ed.), Antibiotics in laboratory medicine. Williams and Wilkins, Baltimore, MD. 2. Davies, J., and D. I. Smith. 1978. Plasmid-determined resistance to antimicrobial agents. Annu. Rev. Microbiol. 32:469-518. 3. Davies, J., M. Cannon, and M. B. Mauer. 1988. Myomycin: mode of action and mechanism of resistance. J. Antibiot. 41:366-372. 4. Hollingshead, 8., and D. Vapnek. 1985. Nucleotide sequence analysis of a gene encoding a streptomycin/spectinomycin adenyltransferase. Plasmid 13:17- 30. 5. Melancon, P., D. Leclerc, L. Brakisr-Gingras. 1990. A deletion mutation at the 5' end of Escherichia coli 16S ribosomal RNA. Biochim. Biophys. Acta 1050:98-103. 6. Ozanne, B., R. Benveniste, D. Tipper, and J. Davies. 1969. Aminoglycoside antibiotics: inactivation by phosphorylation in EScherichie coli carrying R factors. J. Bacteriol. 100:1144-1146. 7. Shaw, R. J., P. N. Rather, R. 8. Hare, and G. H. Miller. 1993. Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes. Microbiol. Reviews 57:138-163. 8. Suzuki, 1., N. Takahashi, 8. Shirato, H. Kawabe, and 8. Mitsuhashi. 1975. Adenylylation of streptomycin by Staphylococcus aureus: a new streptomycin adenylyltransferase, p. 463-473. In S. Mitsuhashi and H. Hashimoto (ed.), Microbial drug resistance. University Park Press, Tokyo. 97 MICHIGAN STATE UNIV. LIBRARIES HHI”WINWNIWWWWWWIWIWW 31293010222606