Nucleotide sequence of the bacterial streptothricin resistance gene sat3

Biochi~ic~a et Biophysica A~ta ELSEVIER

Biochimica et Biophysica Acta 1263 (1995) 176-178

Short Sequence-Paper

Nucleotide sequence of the bacterial streptothricin resistance gene sat3 Erhard Tietze a,*, Jean Brevet

b

a Robert-Koch-lnstitut, Bereich Wernigerode, D-38843 Wernigerode, Germany b lnstitut des Sciences V£gdtales, C.N.R.S., Gif-sur-Yl,ette, France

Received 27 March 1995; accepted 3 May 1995

Abstract

The nucleotide sequence of the sat3 gene which encodes resistance of enteric bacteria to the antibiotic streptothricin is reported. A protein with a molecular mass of about 23 kDa is expressed from this gene. The sat3 gene is not obviously related to any one of the streptothricin resistance determinants identified so far among Gram-negative or Gram-positive bacteria. Keywords: Antibiotic resistance; Streptothricin; sat3 gene; Bacterial plasmid; Incompatibility group IncQ; Nucleotide sequence

Resistance of bacteria to the bactericidal action of streptothricin is due to enzymatic inactivation of the antibiotic by specific acetyltransferases [!,2]. Respective genes were identified in streptothricin-producing streptomycetes [3,4] in Campylobacter coli strains [5] and on a variety of plasmids from enteric bacteria [6]. The recent evolution of St among the intrinsically sensitive enteric bacteria has been attributed to the spread of Tn7-1ike transposons which carry one of the two related streptothricin acetyltransferase genes satl and sat2 [7-9]. Using specific DNA probes, also non-transposing satl or sat2 genes were detected on may different plasmids and also in the chromosome of natural bacterial isolates [10]. However, another St determinant which also codes for a streptothricin acetyltransferase activity but does not hybridize with satl or sat2 gene probes was identified on the plasmid piE693 which belongs to the incompatibility group IncQ [10,11]. The respective resistance gene sat3 was cloned and its nucleotide sequence was determined (Fig. 1). Sequencing of the 1074 bp DNA fragment cloned in

Abbreviations: St, streptothricin resistance; ORF, open reading frame; sat, streptothricin acetyltransferase gene; SAT, streptothricin acetyltransferase protein. The nucleotide sequence data reported in this paper have been submitted to the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number Z48231. * Corresponding author. Fax: + 49 3943 679207. 0167-4781/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 4 7 8 1 ( 9 5 ) 0 0 1 0 3 - 4

plasmid piE1043 (Fig. 2) was performed by the dideoxy chain-termination method [12] after subcloning it into the Sequenest II transposon-deletion-vectors pAA-PZ718 and pAA-PZ719 (GOLD Bio Technology, St. Louis, MO, USA). Overlapping sub-fragments of the insert were generated in vivo by the IS l-mediated random deletion method as recommended by the supplier of the vector plasmids. Routine manipulations of nucleic acids, preparation of single stranded DNA and sequencing reactions using the Sequenase Version 2.0 DNA Sequencing Kit (US Biochemical, Bad Homburg, Germany) were performed as described [ 13]. Computer aided analysis of the sequence presented in Fig. 1 using the facilities of the P C / G E N E program package (IntelliGenetics, Mountain View, CA, USA) reveals an open reading frame spanning from position 221 to 760 (see also Fig. 2) which could be translated to a polypeptide consisting of 180 amino acids. Possible expression signal sequences with good homology to the consensus of conserved motives in Escherichia coli promoters are identified upstream of this ORF (Fig. 1). A sub-fragment lacking the DNA up to the SspI site as cloned in the plasmids piE1044 or piE1045, respectively, is sufficient to mediate St of bacterial host cells (Fig. 2). Therefore, the entire sat3 gene including its natural expression elements is present within this fragment. The resistance phenotypes of host cells harbouring plasmids derived by various deletions into this fragment further

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701 CTGT&'I'~2GTG GC~'I'GC*%TCC GGGC&GCCGA GAGG'I'AGC'I'C T G ~ A 751 TTTGA~A-*-A-A"TAAATGACAA ACTTTGGCCG TCCGGGAAAC GGCACTCGGC 801 CAGATGAGCG GAGTTATGAA TGAGTGAGTG GCGACGACAG CAAGTCTGTG 851 CACCTATTCC CATCd2CTGCG AGCATGGCAA CCAGTCTGGA AGGATACCAA 901 TGGGCGCCTA TCACAATTGG TGAATCCGGC AGCAATGTTT ATCGACTTTA 951 TGGGAAACCA AAAGCTCCTG A'I'"FI%'~'A"I'f'T GAAGCGAGGT AAGTACGACG 1001 TTGCTGATGA TGTGACCGAT GAAATGGTCA GGCTACGCTG GCTTC-CCGAA 1051 CGTATCCCTG TGCCAACCGT CGTC

Fig. 1. Nucleotide sequence of a DNA fragment cloned from the IncQ plasmid pIE639. Small letters represent a sequence also present in the prototype IncQ plasmid RSFI010 [17]. The ORF identified for the St gene sat3 is typed in bold face. Possible conserved elements of an E. coli promoter upstream of the ORF a underlined in addition ( ' - 3 5 region' from position 159 to 164, ' - 1 0 region' from 181 to 186, 'ribosome binding site' from position 208 to 211). Another open reading frame immediately downstream of the putative sat3 gene beginning with ATG a position 820 (double underlined) is not yet completely sequenced.

~ORFsat3~ 221 Pstl Sspl

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1 93

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Plasmid

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localize the St determinant to the region between positions 93 and 787 (Fig. 2). According to these results the ORF identified in the nucleotide sequence represents the structural gene sat3. Selective expression of genes within the insert-fragment of plasmids plE1044 and piE1045 can be achieved from the bacteriophage T7 promoter in the vector DNA (Fig. 2) if E. coli RNA polymerase is inhibited and T7 RNA polymerase is provided [14]. Under respective conditions, a protein with a molecular mass of about 23 kDa is expressed by the plasmid plE1044 (Fig. 3) but not by piE1045 (not shown). This is in good agreement with a value of 20.4 kDa as predicted for a protein translated from ORF sat3 (Fig. 1). Therefore, the protein encoded by the insert of plasmid plE1044 is assumed to represent the streptothricin acetyltransferase SAT3 which give rise to a St phenotype of bacterial host cells. SAT3 is different in

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Fig. 2. Restriction enzyme cleavage map of the IncQ plasmid-derived DNA fragment which contains the St gene sat3. Numbers refer to the nucleotide sequence of this DNA fragment given in Fig. 1. Lines correspond to DNA which is present in plasmids denominated at the right. plE1043 is derived by ligation of a PstI-HindlI fragment of piE693 [11] into the vector plasmid pUC18 [19]. A sub-fragment of piE1043 as generated by digestion with Sspl and EcoRI (which cleaves in the multiple cloning site region of pUC18) was ligated into the expression vector plasmids pT7-5 and pT7-6 [14] to give rise to piE1044 and piE1045, respectively. The orientation of the bacteriophage T7 promoter in the vector plasmid DNA is indicated by arrows. Plasmids piE1046, pIE1047 and pIE1048 were obtained from the recombinant transposondeletion-plasmids mentioned in the text. The endpoints of deletions in these plasmids are defined by sequencing. The sensitivity to streptothricin of bacteria harbouring the respective plasmid is indicated by ' + ' (resistant to 500 /~g/ml) or ' - ' (sensitive to 1 /zg/ml).

Fig. 3. Proteins expressed from the St genes satl, sat2 and sat3 (from left to right). DNA fragments containing the respective resistance genes were cloned in expression vectors to accomplish selective expression under the control of the bacteriophage T7 promoter provided by the vector plasmid [14]. Source of the satl gene was plasmid pOIE38 [8], source of the sat2 gene was plasmid piE935 [11] and expression of sat3 was tested with plasmid pIE1044 (Fig. 2). Labelling of the proteins by incorporation of [35S]methionine, electrophoresis of whole cell proteins in 12% polyacrylamide gels in the presence of 0.1% SDS and autoradiography were performed as described by K~Ssterand Braun [20]. Positions of non-labelled proteins from a molecular weight standard (SDS-PAGE Standards, low range, Bio-Rad Laboratories, Miinchen, Germany) are indicated at the left. The molecular weights of the respective SAT proteins as calculated from their electrophoretic mobilities are indicated at the right. These values are in good agreement with the prediction of polypeptides from translation of the respective ORF in the nucleotide sequences (SATh 56.3, SAT2: 19.7, SAT3: 20.4).

178

E. Tietze, J. Brevet / Biochimica et Biophysica Acta 1263 (1995) 176-178

size from the previously described streptothricin acetyltransferases SAT1 and SAT2 (Fig. 3). Comparison of the sat3 gene with the transposon-determined St genes satl [15] and sat2 [16], with the C. coli St gene sat4 [5] and with the streptomycete St genes star3 nat l [4] does not disclose any significant nucleotide sequence homology. Moreover, concluding from the respective predicted amino acid sequences, the SAT3 protein is not closer related to the enterobacterial SAT1 or SAT2 enzymes than to the St-mediating enzymes of C. coli or streptomycetes [4]. Accordingly, the sat3 gene represents a distinct branch in the evolution of St resistance in bacteria. The natural source of the sat3 gene is the IncQ plasmid piE639. Physical comparison of this plasmid with the prototype IncQ plasmid RSF1010 reveals close similarity except of about 2.2 kb of continuous additional DNA in piE639 [11]. Determinants for resistance of host cells to the antibiotics streptothricin and kanamycin, which are not present in RSF1010, have been located within this additional DNA fragment [11]. The ORF just downstream of the sat3 gene (Fig. 1) probably represents the 5' part of the kanamycin resistance gene of piE639 as concluded from considerable nucleotide sequence homology with the kanamycin resistance gene of the IncP plasmid RP4 [18]. The nucleotides 1 to 130 of the sequence shown in Fig. 1 are the complementary equivalent of the nucleotide sequence from position 7644 to 7773 as determined for RSF1010 [17]. Further sequencing is in progress in order to complete the nucleotide sequence of the whole piece of DNA present in piE639 in addition to the common IncQ plasmid core DNA. E.T. was supported by the 'Fonds der Chemischen Industrie'.

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