Nucleotide sequence homology of the tetracycline-resistance determinant naturally maintained in Bacillus subtilis Marburg 168 chromosome and the tetracycline-resistance gene of B. subtilis plasmid pNS1981

Biochimica et Biophysica Acta, 950 (1988) 441-444

441

Elsevier BBA 90114

BBA Report

N u c l e o t i d e s e q u e n c e h o m o l o g y of the tetracycline-resistance d e t e r m i n a n t n a t u r a l l y m a i n t a i n e d in B a c i l l u s subtilis M a r b u r g 168 c h r o m o s o m e

and the tetracycline-resistance g e n e of B. subtilis plasmid p N S 1 9 8 1 Rciko Sakaguchi, Hitoshi Amano and Kazuo Shishido Department of Ltfe Science, Faculty of Science, Tokyo Institute of Technology, Yokohama (Japan) (Received 18 March 1988)

Key words: Nucleotide sequence homology; Tetracycline resistance determinant; Chromosome; Plasmid; (B. subtilis)

The nucleotide sequence (1579 bp) of tetracycline-resistance determinant and flanking regions of the cloned 5.1 kb DNA fragment from Bacillus subtilis GsYg08 chromosome (Sakaguchi, R. and Shishido, K. (1988) Biochim. Biophys. Acta 949, 49-57) were determined and compared with those of the B. subtilis tetracycline-resistance plasmid pNS1981. The tetracycline-resistance structural (tet) genes of the B. subtilis GSY908 chromosome (tetBSg08) and pNS1981 (tetpNS1981) were found to be Mghly homologous (80% identical). Both tet genes were composed of 1374 bp and 458 amino-acid residues initiating from a GTG codon preceded by a ribosome-binding site (RBS-2). Upstream from tetBSg08 there exists a short open reading frame (20 amino acids) initiating from a ATG eodon preceded by its own RIBS (RBS-1). This leader sequence was also highly homologous to that of tetpNS1981 except for a deletion of one bp between the RBS-I and the ATG codon.

We have recently cloned a 5. lkb DNA fragment from the B. subtilis GSY908 (Marburg 168 strain) chromosome, which confers tetracycline resistance (Tc R) when introduced by transformation into E. coli [1]. This DNA fragment hybridizes strongly to the Tc R region of plasmid pNS1981 from B. subtilis [2]; tetracycline-resistance plasmids indistinguishable from pNS1981 are widely

Abbreviations: kb, kilobase pair(s); bp, base pair(s); RBS, ribosome-binding site; tet, tetracycline-resistance structural gene; Tc R, tetracycline resistance. The sequence data in this paper have been submitted to the EMBL/Genbartk Data Libraries under the accession number GSY908.

Correspondence: K. Shishido, Department of Life Science, Faculty of Science, Tokyo Institute of Technology, Nagatsuta, Yokohama 227, Japan.

dispersed among Gram-positive bacteria, especially aerobic spore-forming bacilli (reviewed in Ref. 3). While the 5.1 kb chromosomal DNA fragment confers significant Tc R on E. coli, this is not always the case in B. subtilis, which shows a strain-dependent Tc R phenotype [1]. In this paper we report the nucleotide sequence of the Tc R structural gene (tet) (and flanking sequences) carried within a 5.1 kb B. subtilis chromosomal DNA fragment and compare this sequence with The Tc R determinant carried by B. subtilis plasmid pNS1981 [4]. The Tc R conferred by the cloned sequence is associated with decreased accumulation of tetracycline into the cells, which is analogous to that conferred by pNS1981 (see Refs. 1 and 5 supporting this claim). According to the strategy shown in Fig. 1, nucleotide sequence of ca. 3.5 kb including the

0167-4781/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

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Fig. l . Sequencing strategy for the Tc R region and neighboring regions on the cloned 5.1 k b fragment from B. subtilm GSY908 chromosome. Arrows indicate the direction and extent of sequence determination. The Tc R region determined by sequence analysis is shown.

expected Tc R region [1] was determined by the method of Sanger [6] and Messing [7]. Just like tetpNS1981 [4], tetBS908 was composed of 1374 bp and 458 amino-acid residues (molecular weight of 50000) initiating from a GTG codon (nucleotides 1-3) preceded by a ribosome-binding site (RBS-2). Both tet genes were found to be significantly homologous (80.2% identical). Upstream from the open reading frame for tetBS908 there

exists a short open reading frame (leader peptide) consisting of 20 amino acids (nucleotides -96(ATG) to -36(TAA)) preceded by its own RBS (RBS-1), being 80% identical with that of tetpNS1981. The synthesis of leader peptide has been considered to be involved in Tc-inducible Tc R in B. subtilis conferred by pNS1981 [3,4]. The Tc R conferred by the cloned sequence of B. subtilis GSY908 chromosome was also Tc-inducible

1

M N T S Y S Q S N L R H N Q I L I W L C ILSFFSVLNE M V L N V S L P D I IIII II II II III I I IIII :11111 :111111111 M N T S Y S Q S T L R H N Q V L I W L C VLSFFSVLNE M V L N V S L P D I

6o ANDFNKPPAS TNWVNTAFML I1: III III :11111111 ANEFNKLPAS ANWVNTAFML

120 TFSIGTAVYG KLSDQLGIKR LLLFGIIINC FGSVIGFVGH SFFSLLIMAR FIQGAGAAAF II III :11 I I I I I I I I I IIIII1:II I1:11 II III;II1:11 IIII II1! TFSIGTALYG KLSDQLGIKN LLLFGIMVNG LGSIIGFVGH SFFPILILAR FIQGIGAAAF 180 PALVMVVVAR YIPKENRGKA FGLIGSIVAM GEGVGPAIGG MIAHYIHWSY LLLIPMITII II III II IIIIII II IIIIIIIIII ifitI It IIIIIIIIII IIII I:111 PALVMVVVAR YIPKENRGKA FGLIGSLVAM GEGVGPAIGG MVAHYIHWSY LLLIPTATII 240 TVPFLMKLLK KEVRIKGHFD IKGIILMSVG IVFFMLFTTS YSISFLIVSV LSFLIFVKHI II I lllI:l: I:I III I II I1: II II I1:11 I : II III I IIIIII TVPFLIKLLK KEERIRGHID MAGIILMSAG IVFFMLFTTS YRFSFLIISI LAFFIFVQHI 300 RKVTDPFVDP GLGKNIPFMI GVLCGGIIFG TVAGFVSMVP YMMKDVHQLS TAEIGSVIIF I II II I II I:111 IIIIII II IIll III I I I! It[ It III1:I:1 RKAQDPFVDP ELGKNVFFVI GTLCGGLIFG TVAGFVSMVP YMMKDVHHLS TAAIGSGIIF 360 PGTMSVIIFG YIGGILVDRR GPLYVLNIGV TFLSVSFLTA SFLLETTSWF MTIIIVFVLG I IIII II III1:1111: I:11 I II I II : I IllIl:l[ PGTMSVIIFG YIGGLLVDRK GSLYVLTIGS ALLSSGFLIA AFFIDAAPWI MTIIVIFVFG 420 G L S F T K T V I S TIVSSSLKQQ E A G A G M S L L N F T S F L S E G T G IAIVGGLLSI P L L D Q R L L P M II IIII II I:111111: IIII IIIII IIIIII II IIIIIIII1! : if f i l l : GLSFTKTVIS TVVSSSLKEK EAGAGMSLLN FTSFLSEGTG IAIVGGLLSI GFLDHRLLPI 458 E V D Q S T Y L Y S N L L L L F S G I I V I S W L V T L N V YKHSQRDF--teCpNS1981 :ll lJl l! I : 1 : 1 1 1 1 1 1 II I I III I1:1 I D V D H S T Y L Y S N M L I L F A G I I V I C W L V I L N V YKRSRRHG--tetBS908

Fig. 3. Homology of the deduced amino-acid sequences of the TET proteins of tetpNS1981 and tetBS908. Marks (]) a n d (i) are

identical and favored amino-acid residues [9], respectively.

443

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AATTAAAAGACCGAAGCGTATC .UTTMA.+GAGGGA4GC~TATC

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TTGTTT TTGTTT

ATGACAATTAT ATGACAATT4T

TCA TCA

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(SGAAG FGAAG

GCTTTTTATCAGAGGGM GCTTTTTATCAGaGGGAA

CTGGAATGACTTTGCTT CTGGAATGAGTTTGCTT

1320 AGCAATCATT ACGAATCATT fCA-tetpNS1981 JiTC-tetBS908

Fig. 2. Nucleotide sequences of the TcR regions of pNS1981 (upper) and B. subrilis GSY908 chromosome (lower), numbered from the GTG initiation codons of both ter genes. Identical bases between the two sequences are boxed. Translational start codons and stop codons of the TET protein and leader peptide and putative ribosome-binding sites (RBS) are shown. putative promoter and mRNA start point of rerpNS1981 are also shown. The arrow ( fi) shows the deletion point of rerBS908. The point indicated is a Dots are positioned every 10 bp.

putative point. The following arrangement is also possible: ~@++@~I. ”

444

in B. subtilis [1]. It was, however, noteworthy that the leader region of tetBS908 lacks one basepair between the RBS-1 and the A T G initiation codon when compared with that of tetpNS1981. The RBS-1 of tetpNS1981 leader perfectly matches the 3'-terminal sequence of B. subtilis 16S ribosomal R N A (HoUCUUUCCUCCACUAG---), while one G-U pairing occurs in the RBS-1 of tetBS908 leader. The transcriptional start point of tetpNS1981 has been estimated at 10 bp upstream from the RBS-1 [4,8]. 8 out of the 10 bp were identical between tetBS908 and tetpNS1981. While the sequence upstream from the deduced m R N A start point was not so homologous between both tet genes. Similarly, a significant homology was not observed in the sequence downstream from the TAA stop codon (nucleotides 1375-1377) of the open reading frame of tet gene, although a small portion of the sequence for each gene is shown in the figure. Both T E T proteins deduced show a high homology (81.4% identical) and are highly hydro-

phobic, as expected for a membrane-bound protein [1,5] (Fig. 3). This work was supported in part by a Grant-in Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. References 1 Sakaguchi, R. and Shishido, K. (1988) Biochim. Biophys. Acta 949, 49-57. 2 Shishido, K., Noguchi, N., Kim, C. and Ando, T. (1983) Plasmid 10, 224-234. 3 Shishido, K. (1987) Seikagaku 59, 74-81. 4 Sakaguchi, R., Shishido, K., Hoshino, T. and Furukawa, K. (1986) Plasmid 16, 72-73. 5 Sumita, Y. and Shishido, K. (1985) FEMS Microbiol. Lett. 30, 403-406. 6 Sanger, F. (1981) Science 214, 1205-1210. 7 Messing, J. (1983) Methods Enzymol. 101, 20-78. 8 Hoshino, T., Ikeda, T., Tomizuka, N. and Furukawa, K. (1985) Gene 37, 131-138. 9 Dayhoff, M.D., Schwartz, R.M. and Orcutt, B.C. (1978) in Atlas of Protein Sequence and Structure (Dayhoff, M.D., ed.), Vol. 5, Suppl. 3, pp. 345-352.