IS257 from Staphylococcus aureus: member of an insertion sequence superfamily prevalent among Gram-positive and Gram-negative bacteria

195

Gene, 76 (1989) 195-205 Elsevier GEN 02867

IS257 from Stuphylococc~~ awezw member of an insertion sequence superfamily prevalent among Gram-positive and Gram-negative bacteria (Transposase; structure;

evolutionary

immigrant

comparisons;

codon

bias;

G + C content

selection;

protein

evolution;

genetic

genes)

Duncan A. Rouch and Ronald A. Skurray Department of Microbiology, Monash University, Clayton, Victoria 3168 (Australia) Received

by P.A. Manning:

Accepted:

5 September

28 June 1988

1988

SUMMARY

The nucleotide sequences for the IS257 family of insertion sequences from Staphylococcus uureus were compared with those of the ISSl family from Streptococcus Zactis and the IS15 family which is widespread amongst Gram-negative bacteria. These elements have a striking degree of similarity in both their putative transnosase polypeptide sequences and their nucleotide sequences (40 to 64% between pairs), including 12 out of 14 bp conservation in their terminal inverted repeats. The evolutionary distance between the IS15 family and the IS257 and ISSl families of Gram-positive origin is approximately twice that between the IS257 and ISSl families. Analysis of base substitutions in the three sequences has provided insights into the effect of selection for the G + C content of immigrant genes to conform to that of their hosts, and into the evolution of biases in overall amino acid composition of cellular proteins in prokaryotes and eukaryotes. The IS257, ISSI, IS15 families form a superfamily of insertion sequences that has been involved in the spread of a number of antimicrobial resistance determinants in Gram-positive and Gram-negative pathogens.

INTRODUCTION

are thought to have played a pivotal role in the emergence of multi-drug resistance in pathogenic bacte-

Transposable elements, through their ability to enhance gene transfer and rearrangements via homologous and non-homologous recombination processes and to affect gene expression (Cohen, 1976; Campbell, 1981; Kleckner, 1981; Syvanen, 1984),

ria, such as S. uureus (Lyon and Skurray, 1987; Skurray et al., 1988) and the streptococci (Clewell, 198 1; Clewell and Gawron-Burke, 1986). One such element from S. aureus is IS257, of which directly repeated copies flank the trimethoprim-resistance determinant on many members of the pSK1 family of multi-resistance plasmids, forming the composite transposon Tn4003 (Gillespie et al., 1987; Lyon and Skurray, 1987; Rouch et al., 1989). IS257 also forms direct repeats bounding mercury-resistance determinants on heavy-metal-

Correspondence to: Dr. R.A. biology,

Monash

University,

Tel. 61-3-5654927; Abbreviations: repeat(s);

Skurray, Clayton,

Department Victoria

of Micro-

3168 (Australia)

Fax 61-3-5654007.

aa, amino acid(s);

IS, insertion

sequence(s);

0378-l 119/89/%03.50 0 1989 Elsevier

bp, base pair(s);

IR, inverted

nt, nucleotide(s).

Science Publishers

B.V. (Biomedical

Division)

196

resistance plasmids to constitute the putative transposable element Tn4004 (Gillespie et al., 1987; Lyon and Skurray, 1987); these repeats associated with mercury resistance on the latter plasmids are also known as IS432 (Barberis-Maino et al., 1987). IS257 sequences are additionally located adjacent to chromosomal determinants for resistance to mercury, methicillin and tetracycline (Gillespie et al., 1987; Matthews et al., 1987), and may be involved in the chromosomal integration of these three determinants and the in vitro amplification of the methicillin-resistance determinant (Gillespie et al., 1987; Matthews and Stewart, 1988). Furthermore, up to live copies of sequences homologous to IS257 have been identified on aminoglycoside-resistance plasmids from North America (Gillespie et al., 1987); two of the IS257-like elements on these plasmids form part of the inverted repeats which flank the aacA-uphD aminoglycoside-resistance determinant (M. Byrne, D.A.R., M. Gillespie and R.A.S., in preparation). Remarkably, the IS257 family of insertion elements, of which six members have been sequenced (Barberis-Maino et al., 1987; Rouch et al., 1989) shares sequence homology with the IS15 family which is widespread among R plasmids in enterobacteria, and is also detected in Acinetobacter and Cumpylobacter-like species (Lab&e-Roussel et al., 198 1; Labigne-Roussel and Courvalin, 1983; Ouellette et al., 1987); it includes IS15, ISIS-d (Labigne-Roussel et al., 1981; Trieu-Cuot and Courvalin, 1984), IS26 (Mollet et al., 1983), IS46 (Brown et al., 1984; Hall, 1987) and IS140 (Brau and Piepersberg, 1983). ISSI, detected on a lactose plasmid from S. luctis (Polzin and Shimizu-Kadota, 1987), is also homologous to IS257 (Murphy, 1988; Rouch et al., 1989). We present a detailed sequence comparison of members of the IS257, ISSl and IS15 insertion sequence families, to examine the evolutionary relationships of this collection of IS elements.

MATERIALS (a)

AND METHODS

Sources of nucleotide sequences and their align-

ment

The sequences included were of IS257L, IS257R1, IS257R2 (Rouch et al., 1989) IS43IL,

IS431 R, IS43lmec (Barberis-Maino et al., 1987) ISSl S, ISSlT (Polzin and Shimizu-Kadota, 1987) in the ISSl family; ISIS-& ISIS-d11 (Trieu-Cuot and Courvalin, 1984), ISIS-d111 (Ouellette et al., 1987) and IS26R (Mollet et al., 1983) in the IS15 family. IS46 and IS140 were not included in the analysis as only limited sequence information is available for them (Hall, 1987; Brau and Piepersberg, 1983). Sequence alignment was performed using the programs of Staden (1986) as modified by A. Kyne, Walter and Eliza Hall Institute for Medical Research, Melbourne, Australia. To optimize alignment in the coding region, the putative transposase polypeptide sequences were aligned using the mutation data matrix (Schwartz and Dayhoff, 1978) with a deletion penalty of 8 and a zero matrix bias; the corresponding nucleotide sequences were then positioned according to the polypeptide alignment. This procedure was applied because of the moderate degree of similarity between the nucleotide sequences from the different families. (b) Evolutionary relationships

Phylogenetic relationships were determined with the parsimony analysis of Fitch (197 l), utilizing the nucleotide sequence alignment shown in Fig. 1. To delineate the main branches in the evolutionary tree, representative sequences from the three IS families were analysed; namely, IS257L, ISSlS and ISIS-d& for the IS257, ISSl and IS15 families, respectively. (c) Codon bias

The reading frame for each sequence was scored for the degree of use of the codons preferentially utilized by highly expressed genes, corresponding to the major isoaccepting tRNA species in Escherichia coli, and Bacillus subtilis, according to Bennetzen and Hall (1982). The preferred codons for E. coli and B. subtilis, taken from Sharp and Li (1986) and Shields and Sharp (1987) respectively, were UUC, CUG, GUU, GUA, UCU, UCC, CCG, ACU, ACC, UAC, CAC, CAG, AAC, AAA, GAC, GAA, CGU and GGU for E. coli, and UUC, UUA, CUU, AUC, GUU, UCU, CCU, ACU, ACA, GCU, UAC, CAU, CAA, AAC, AAA, GAC, GAA, CGU, GGU and CGA for B. subtilis. Codon bias is

197

equal to the number of preferred codons used divided by the corrected total number of codons, which is the total codon number minus the number of codons utilized for methionine, cysteine, tryptophan and termination, with alanine codons additionally excluded for E. coli only.

RESULTS AND DISCUSSION

(a) Evolutionary

relationships

among

the IS257,

ISSl and IS15 families

To initiate examination of the evolutionary relationships among members of the IS257, ISSl and IS15 families, their nucleotide sequences, where available, were aligned (Fig. l), as were the polypeptide sequences of their putative transposase enzymes (Fig. 2). The DNA and polypeptide sequence similarities between representatives of the three IS families, viz., IS257L, ISSl S and ISI5-d1, derived from these sequence alignments are shown in Table I; the simil~ti~s leave no doubt that the sequences are related and that IS257L and IS52 S are the most closely related pair. Construction of a phylogeny allows for a more accurate and detailed analysis of the evolutionary relationships between these sequences and the IS families they represent. In dete~i~ing the phylogenetic relationships, 369 positions out of 842 in the nucleotide sequence alignment were informative with regard to differentiating substitutions; at 119 nt positions each sequence contained a unique nt, and there were 14 insertionldeletion regions, shown by gaps in the TABLE I Sequence similarities between IS257L, ISSl S and ISIS-d1 O/,homologies a

IS257L ISSl s ISIS-d1

lS257L

ISSlS

ISIS-AI

(100) 59 40

64 (100) 46

49 50 (100)

a Percentage nucleotide sequence identities are shown above the diagonal and amino acid sequence identities below. These are derived from the nucleotide sequence alignment (Fig. 1) and the corresponding amino acid sequence alignment (Fig. 2).

aligned sequences (Fig. 1). The distance score for each sequence was calculated for its branch in the unrooted tree (Fig. 3a). Assu~~g uniform mutation rates, the rooted evolutionary tree shown in Fig. 3b is produced; remarkably, the distance scores for IS257L and ISSZ S from their last common ancestor are very similar (167 and 161, respectively), supporting the ass~ption regarding mutation rates. The distance between ISI5-BI and IS257L (649) or ISSlS (643) is approx. two-fold greater than the distance between IS257L and ISSlS (328). Compared to the distance between the prototype of each family, the distances within each family are quite small, as suggested by inspection of Figs. 1 and 2, being less than 12 for the most divergent pair, IS257Rl and IS43ZL (not shown). Thus, each family forms a tight cluster, which is consistent with the notion that since their initial divergence, elements from the three families have emerged relatively recently in staphylococci, streptococci and enterobacteria; in these cases the elements have emanated from at least three separate sources, that are likely to be non-pathogens of soil origin. In the case of the IS257 family, S. aureU.sappears to have acquired copies of this IS element in at least two sets: IS431 L and IS431 R, which flank the merA merB mercury-resistance determinant on heavy metal/b-lactamase plasmids (Barber&Main0 et al., 1987; Gillespie et al., 1987), show significant sequence divergence from the IS257L, IS257Rl and IS257R2 sub-families (distance = 10 to 1l), that are components of the trimethoprim-resistance transposon Tn4003 found on quaternary ammoniumcompound-resistance plasmids (Gillespie et al., 1987; Skurray et al., 1988, Rouch et al., 1989). The IS431 pair may have been first selected in nosocomial staphylococci with the advent of mercurial antiseptics late last century, whereas Tn4003 was not detected until 1979, subsequent to the introduction of t~ethoprim in chemo~erapy (Lyon and Skurray, 1987). In contrast, divergence among most members of the IS15 family that have been sequenced is minimal, supporting the contention that the widespread occurrence of elements in this family is largely due to recent intra- and inter-species genetransfer events between Gram-negative pathogens, promoted indirectly by antimicrobial chemotherapy (Lab&e-Roussel and Courvalin, 1983; Ouellette et al,, 1987).

198

-35 I

-10

RBS

MRYFRYKPF

IS257L/R2 ISZR; IS431L ISgR 1s431mec

GGTTCTGTTGCAAAGTTGATTATAGTATAATTTTAACAA?= GGAGTCTTCTGTATGAACTATTTCAGATARACAATTT ................................................................................... ............................................. ..A ................................... .A ................................................................................. ....................... ..R .........................................................

ISSlS ISSiT

. . . . . . . . . . . . . . . ..TTCCGATA...C..T...AGTGT....TGAATARAAATGACAGC.AG.ATA.A.CA...........T.A.GG.......... . . . . . . . . . . . . . . . ..TTCCGATA...C..T...AGTGT....TGAATARAnATGAU\GC.AG.ATA.A.CA...........T.A.GG..........

-35

-

I

-10

RBS

-10

-35

RBS

ISl5-AI IS-i&AI1

..CA..........TAGTCGG.GG.UI..A.C..A.C.TCCCCTT..........?TGCTGATG.AGC.GCAC....,.CCA....A.GGCCGG..T... ..CA..........TAGTCffi.GG.GA..A.C..A.C.TCCCCTT..........~GCTGATG.AGC.GCAC......CCA....A.GGCCGG..T...

IS26R -

..CA..........TAGTCGG.GG.GA..A.C..A.C.TCCCCTT..........~GCTGATG.AGC.GCAC......CCA....A.ffiCCGG..T...

IS257L/RZ IS257Rl IS431L ISCR IS431mec ISSlS ISSiT ISl?-AI IS15-AI1 ISE-AIII IS26R

-

100

20 40 NKDVITVAVGYYLRYALSYRDISEILRGRGVNV ~CAAffiATGTTATCACTGTAGCCGTTGGCTACTACTAnGCGTTC 200 .. .. .. .. .. . ... .. . .. .. . .. .. .. .. .. . .. .. .. .. . . ... . ... ... . . ... . .. .. . .. ... .. .. .. .. .. .. .. .. .. .. .. ... .. .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..A................. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..A................. . . ..R...............*........................A....................................A................. C.A..A.....G........C..T.....T..T......C.T...AATC.A..C........A..TCAA...C.CC.~A...T.....CA.T......T C.A..A.....G........C..T.....T..T......C.T...AATC.A..C........A..TCRA...C.CC.2TA...T.....AA....T...T C..A..G..T..CG C.GCGT..CA.C...C.GTGG.....AC...GG..CTGC.A...C.GCA.C.....C.....GC.GCAG..G..GC.GGCT.A... C.GCGT..CA.C...C.GTGG.....AC...GG..CTGC.A...C.GCA.C.....C.....GC.CCAG..G..GC.ffiCT.A...C..A..G..T..CG C.GCAG..G..GC.ffiCT.A...C..A..G..T..CG C.GCGT..CA.C...C.GTGG.....AC...GG..CTGC.A...C.GCA.C.....C.....GC.GCAG..G..GC.GGCT.A...C..A..G..T..CG 60 HHSTVYRWVQ

EYAPILYQ

I

ATCATTCAACGGTCTACCGTTGGGTTCAAGAATATGCCCCAATTTTATATCAA .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

IS43lL IS43lR 1s43imec ISSlS IssiT ISI?-AI IS%.-AI1 ISE-AI11 IS26R

. . ..C..C...A.T.....C........GCGT.....G..TGAAA.GG.AA..CffiC.GCGC...T.CTGGCGTA.CCCTTCC.A.CT..G.CCG....A . . ..C..C...A.T.....C........GCGT.....G..TGAAA.GG.M..CGGC.GCGC...T.CTGGCGTA.CCCTTCC.A.CT..G.CCG....A

IS257L,'RZ IS257Rl IS43lL IS43lR

ATT

WKKKHKKAYYKWR GCTTATTACAAATGGCG 300 TGGAAGAAAAAGCATAAAAAA ... ... . ... .. . .. .. .. .. .. ... .. .. .. .. ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..A .. .. ... . ... .. . ... .. . ... .. . .... .. . .. ... . .... .. . ... .. . .. .. ... .. .. ... . ... .. .. .. . . . ..A..G..AA...G.C.GT.C.TC..?TCG...AA . . . ..A..G..AA...G.C.GT.C.lC..TTCG...AA

IS257L/R2 IS257Rl

.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..G...... ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..G...... .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..G...... .. . G...CA....TA.T..T........A........CAGTAA.G.CC.C.....T C.C G...CA....TA.T..T........A........CAGTAA.G.CC.C.....T C.C . . ..C..C...A.T.....C........GCGT.....G..TGAAA.GG.AA..CGGC.GCGC...T.CTGGCGTA.CCCTTCC.A.CT..G.CCG....A T.CTGGCGTA.CCCTTCC.A.CT..G.CCG....A . . ..C..C...A.T.....C........GCGT.....G..TGRAA.GG.RA..CGGC.GCGC...

100 SO IDAEGHTLDIWLRKQ IDETYIKIKGKWSYLYRA TATTGATGAGACGTACATCAT~G~T~AGCTATTTATATCGTGCCATTGATGCA~~GACATACATTA~TATTTGGTTGCGT~GC~

400

. .. .. .. . ... .. .. .. .. . .. . .. .. .. . .. .. . .. .. . . .. .. . .. .. .. ... . . ... .. . .. .. .. .. .. .. .. .. .. .. . .. ... . .. .. ... .. . . . ... .. . ... ... . .. .. .. . .. .. ... . .. .. . .. .. . . .. .. . ... .. . ... . .. ... . . .. .. .. .. . ... .. .. .. .. . .. ... .. . ... .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..r.................................

IS43lmec

....................................................................................................

ISSlS

A..G.....A..T..T........C.....TCGT...CAT...C

ISSIT ISE-AI ISIS-AI1 ISIS-AI11

. . . . . . . .A........G..T...?TG..T.....C..C...C.A..C...A.. A..G.....A..T..T........C.....TCGT...CAT...C.C ATC.CTCCTCC.GT C..G.....A..C...G.G..GG.C..T..CCGC...GCG...C.G..C..G...G.C..CAGCCG...C.GC..TG.C...T... C..G.....A..C...G.G..ffi.C..T..CCGC...GCG...C.G..C..G...G.C..CAGCCG...C.GC..TG.C...T...ATC.CTCCTCC.GT C..G.....A..C...G.G..ffi.C..T..CCGC...GCG...C.G..C..G...G.C..CAGCCG...C.GC..TG.C...T...ATC.CTCCTCC.GT C..G.....A..C...G.G..GG.C..T..CCGC...GCG...C.G..C..G...G.C..CAGCCG...C.GC..TG.C...T...ATC.CTCCTCC.GT

IS26R

.C ........ A........G..T...TTG..T.....C..C...C.A..C...A

140

120 RDNHSAYAFI

K R L AAACGTCTC . .. . .. . .. ... . .. .. . . .. .. . .. .

IKQFGKPQKVITDQAPS ATTAAACAATTTGGTAAACCTCAAAAGGTC 500 .. ... .. . .. .. . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .... ... .. .. ... .. .. .. . .. . .. .. .. .. .. .. .. .. .. ... . .. .... . .... ... .. .

.........

. .. ... . . ... .. . . .. .. .. ... .. .. ... .. .. . ... ... . .... ... . .

IS257L/R2 IS257Rl IS431L IS43lR

CGAGATAATCATTCAGCATATGCGTTTATT . . . . . . . . . . . . . . . . . . . . . . . . . . . ..c

IS43lmec

. . . . . . . . . . . . . . . . . . . . . . . . . . . ..c

ISSlS

. . . ..A..A ..GA...CG..AG.T..T........CT.G . . . ..A..A ..GA...CG..AG.T..T........CT.G ..TA.C.GCA.AG.T.....CCG....C.GGGT...ATC... ..TA.C.GCA.AG.T.....CCG....C.GGGT...ATC... ..TA.C.GCA.AG.T.....CCG....C.GGGT...ATC... ..TA.C.GCA.AG.T.....CCG....C.GGGT...ATC...

-

ISSiT ISly-AI ISC-AI1 IS'5-AI11 IS26R

. . . . . . . . . . . . ..G..............C . . . . . . . . . . . . ..G..............C

..

... ... . .. .. . .. .. . .. .. .. .. ... .. ... .. . .. ... ... .. .... ..

CA......G......C....AAG.GTAA.TG.C..G...A.A..G..C..'r. CA......G......C....AAG.GTAA.TG.C..G...A.A..G..C..T. AACAACG.G..GA.G.W;CAG.TC..A.G.TTCA.C.AC..G...A.A..G..CG.CT AACAACG.G..GA.G.GGCAG.TC..G.G.TTCA.C.AC..G...A.A..G..CG.CT AACAACG.G..GA.G.ffiCAG.TC..G.G.~CA.C.AC..G...A.A..G..CG.CT ARCAACG.G..GA.G.ffiCAG.TC..G.G.TTCA.C.AC..G...A.A..G..CG.CT 160

TKVAMAKV IS257L/R2 IS257Rl -

CGAAGGTAGCAATGGCTAAAGTA . . . . . . . . . . . . . ..T.......

IS431L IS43lR IS43lmec

.. . .. ... .. .. ... .. .. .. .. . . . . . ..I...............

IKAFKLKPDC ATTAAAGCTTTTAAACTTAAACCTGACTGC

HCTSKYLNNLIEQD CATTGTACATCGAAATATCTGAATAACCTCATTGAGCAAGA

. ... . .. .. . .. .. . .. ... . .. ... .. ..

. . ... . .. .... . ... .. .. .. .. .. . ... . .. ... . .. .. .. . .. .. .. ... .. .. ... . .. .. .. .. .. .. . ... .. . .. .. . .. ... .. ... .. . ... . ... .. . .. .. .. .. .. .. . ..

. . . . . . . . . . . . . . . . . . . . . . . . . . . ..T . . . . . . . . . . . . . . . . . . . . . . . . . . . ..T

....................................................

.

..G

.........................................

. ..C.A..CGT...G.....C

....................

ISSlS

TTGGTTCT...T.TAGR..GT..CAG.G...C.G...AT.TAC...GR.A

ISSlT IS-AI

. ..C.A..CGT...G.....C.................... TTGGTTCT...T.TAGA..GT..CAG.G...C.G,..AT.TAC...GA.A..G .AACGC.MffiCCGGTGCCCGT.....GTTGRA..CCAACAGRTT..G..C.G...C...G.G.....ATGC.. ATGGTCGC..GC.T...CTGC.C

ISIS-AI1 ISlS-AI11 IS26R -

ATGGTCGC..GC.T...CTGC.C ATGGTCGC..GC.T...CTGC.C ATGGTCGC..W:.T...CTGC.C

.AACGC.AAGGCCGGTGCCCGT.....GTTGAA..CCAACAGATT..G..C.G...C...G.G.....ATGC.. .AACGC.AAGGCCGGTGCCCGT.....~TGAA..CCAACAGATT..G.GC.G...C...G.G.....ATGC.. .AACGC.AAGGCCGGTGCCCGT.....~TGAA..CCAACAGATT..G..C.G...C...G.G.....ATGC..

600

199

200

180 ISEL/RZ IS257Rl IS431L IS431R xs431mec ISSlS ISSlT _ ISE-AI ISE-AI1 ISlS-AI11 -_ IS26R

H R H I K TCACCGTCATATTAAA .. . .. . ... . .. .. .. .. ... .. ... . .. ... .. .. . ... . .. .. .. . .. .. . .. .. .. .. ... C..T..A.CA..C...

V R K GTAAGAAAG .. . .. .. . . .. . .. .. . . .. . . .. .. . .. . . .. .. . CG.C.C..T

C..T..A.CA..C...

CG.C.C..T

. ..TG.CA.AC.G...CGGA...TC..CGCC..

YQSINTARNTLKGIECIYAL TATCAAAGTATCAATACAGCAAAGAATACT"TTAUAGGTATTGAATGTATTTACGCTCTA ACAAGG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..G............... . .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..A..... ... .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..A..... ... .. . . .. .. . .. ... . .. .. .. .. .. . .. ... .. . ... . .. .. .. .. .. .. .... .. .. ... .. . .. .. . .A.TTT . . ..G.... C.ACGA..T..CTCA.CC..GA.T..G..C..G...ACA...CGA.GAA.. . . ..G....C.ACGR..T..CTCA.CC..GA.T..G..C..G...ACA...CGA.GAA.. .A.TTT CCT.CGA.T.A..TCC..G..G..G..TT.CGCC..CR.CGT..A... T

R

TCGGCGCC..GCT.GGA.T.A..TCC..G..G..G..~.CGCC..CA.C...........GGTG..GCGT..A... . ..TG.CA.AC.G...CGGA... . ..TG.CA.AC.G...CffiA...TCffiCGCC..GCT.ffiA.T.A..TCC..G..G..G..TT.CGCC..CA.C...........ffiTG..GCGT..A... GCT.GGA.T.A..TCC..G..G..G..~.CGCC..CA.C...........GGmr..GCGT..A... . ..TG.CA.AC.G...CGGA...TCGGCGCC.. 220 YKKNRRSLQI YGFSPCHEISIMLAS* TATAAAAAGAACCGCAGGTCTCTTCAGATCTACGGATTTTCGCCATGCCRCWUGCG ................................................................................................. ................................................................................................. .................................................................................................

IS257L,'R2 ISERl IS431L IS431R IS431mec ISSlS ISSLT IS=-AI ISE-AI1 ISl5-AI11 IS26R

/ ACTTTGCAACAGAACC

ATTAGTGGTTAGCTATATTTTTT

IS15-AI1 IS15-AI11 IS26R

GAC.AAA.GCT..CTC..CGC GAC.AAA.GCT..CTC..CGC

.A .A

GAC.AAA.GCT..CTC..CGC

.A

842

................ . ....................... ...................................... . ....................... TA..AACC..GTA.T.GA ..... AA TA..AACC..GTA.T.GA ..... AA GAC.AAA.GCT..CTC..CGC .A

Nucleotide

sequence

alignment

................ ................ ................ .......... ..TG .. .......... ..TG .. .......... ..TG .. .......... ..TG ..

for members

of the lS257, ISSl

for lS257L, and lS257R2 (lS257L/R2);

for the other aligned sequences,

Bold lines mark the positions

promoter

of each family. Alignment well conserved lS257L/R2 according

among

to facilitate

of putative position

members

numbers

alignment.

unknown.

nucleotides

sequence

Uncertainties

is shown

sequence

and ISIS-All1

sites (RBS) for the members indicate

numbers

(Barberis-Maino

sequences

the 14-bp IR which is

for the transposase

(Fig. 2); associated

in the lS43lmec

Note that the lS431mec

ribosome-binding

lines at the termini

Translation

alignment

sequence is shown

nucleotide

are only shown where they differ from lS257L/R2.

(-35 and -10) and potential

ISSZ and ISIS families.

with the polypeptide

sequence

and by an asterisk,

sequences

and IS.15 families. The complete

are shown to the right. Half-arrowed

of the lS257,

orientation

to the polypeptide

by R, purine,

800

............................... ..C...........A..AAA.GGRAC.C...T..........GT..CTACT..G....AGG..T.AA.G.G..TA.T T.AGAAC.AGAAGG.T . ..T ..C...........A..AAA.GGAAC.C...T..........GT..CTACT..G....Affi..T.RA.G.G..TA.T T.AGAAC.AGAAGG.T . ..T CGC..AGG..AGGCC..AGCATTTTAT..T ..T GAT..CCTGGG......GC..C.ffi.AAGCAG.DT..TT.AAATGT.AffiCCTTTGA .... CGC..AGG..AGGCC..AGCAWTTAT..T ..T GAT..CCTGGG......GC..C.GG.RAGCAG.GP..TT.AAACGT.AffiCCTTTG8 .... CGC..AGG..AGGCC..AGCATTTTAT..T ..T GAT..CCTGGG......GC..C.GG.MGCAG.GP..TT.AAACGT.AGGCCTTTGA .... CGC..AGG..AGGCC..AGCATTTTAT..T ..T GAT..CCTGGG ...... GC..C.GG.AAGCAG.GT..TT.AAATGT.AGGCCTTTGA ....

IS257L,'R2 IS257Rl ISHL IS431R ISSlS ISSiT IS~-AI

Fig. 1.

AACACTGRCATGATAA

reading

thus indicate

frame

of

positions

et al., 1987) are indicated

are incomplete

at the 3’ and 5’ ends,

respectively.

(b) Codon usage within transposase genes Codon

usage within

a gene reflects both on the

identity of the host that it originated from (Grantham et al., 1980; Bibb et al., 1984) and on the degree to which it is expressed; the more highly expressed a gene is the greater the use of synonymous codons corresponding to the major isoaccepting tRNA species within the host with which it has had a longterm association (Post et al., 1979; Bennetzen and Hall, 1982). The level of use of these preferred codons in the putative transposase genes located in the three prototype IS elements, IS257L, ISSl S and ISIS-d1 (Fig. 1, cf. Fig. 2), was measured for representative Gram-positive and Gram-negative host species, namely B. subtilis and E. coli, respectively, using the codon bias statistic of Bennetzen and Hall (1982), which allows discrimination between dif-

ferent hosts. The codon bias results (Table II) show that the two sequences of Gram-positive origin, IS257L TABLE Codon

and ISSIS,

have a higher codon

bias for

II bias in transposase

Host

Insertion

genes sequence

lS257L Codon

ISSl s

ISlS-Al

bias ’

B. subtilis

0.45

0.42

0.34

E. coli

0.37

0.35

0.54

a Codon bias was calculated METHODS,

section

levels show a codon 1982).

c. Genes

according expressed

to MATERIALS

AND

at low and moderate

bias of less than 0.6 (Bennetzen

and Hall,

200

20 IS257L,'RZ IS257Rl IS431L

MNYFRYKQFNKDVITVAVGYYLRYALSYRD MNYFRYKQFNKDVITVAVGYYLRYALSYRD MNYFRYKQFNKDVITVAVGYYLRYALSYRD

I/// Helix ///I--Tff"-I///// Helix /////I 60 ISEILRGRGVNVHHSTVYRWVQEYAPILYQI-ml-IWKKKHKKAYYKW] I SE I I, RGIIJ R GV N VHLI S TV Y RW VQ E Y A P I L Y Q - I -W K K K iIK K A Y Y KW IS EILRER G V N V II II S TV Y ~W VQ E Y A P I L Y U - 1-W K K K ii K K A Y Y KW ISEILRER GVNVHHSTVYRWVQEYAP I LYQ- I /II -WKKKHKKAYYKW I ISEILRERGVNVHHSTVYRWVQEYAPILYQ-u-WKKKHKKAYYKW IQE~LYiJRGLNVCH~T~YRWVQ&YSK~LY~-k-WKKKfNKQSEYSh NVDHSTIYRWVQRYPEMEKRLRWYWRNPSDLCPW @jL-fRGg 1 [ [ ~!++=j LQEMLAERGVNVDHSTIYRWVQRYAPEEEKRLRWYWRNPSDLCPW LQEfiLAERGVNVDHSTrYRWVQRYAPEgEKRLRWYWENPSDLCPW LQEijLAERGVNVDHSTiYRWVQKYAPEGEKRLRWYWBNPSDLCPW

IS257L/R2 ISmRl IS431L IS43LR ISZXmec ISSlS/T ISl%-AI ISi?-AI1 ISi?-AI11 ISSR

I

l/// Helix ///I-T"f"--[///// Helix /////I IS257L/R2 ~RIDETYIKIKGKW~YLYRAIDAEGHTLUIWLRKQRDNHSAYAFI~ISmRl ISz-lL IS431R IS431mec ISSlS/T ISl5-A I ISi%-AI1 ISi-!?-AI11 ISZR -

100

120

IS257L/RZ IS257Rl IS431L ISmR IS431mec ISSlS/T ISl?-A I ISi??-AI1 IS=-AI11 IS?%R -

IS257L/R2 ISmRl ISB-iL IS431R 1szimec ISSlS,'T ISls-AI ISi%-AI1 ISZ-AI11 IS%R 220 KKNRRSLQIYGFSPCHEISIMLAS KKNRRSLQIYGFSPCHEISIMLAS KKNRRSLQIYGFSPCHEISIMLAS

IS257L/R2 ISxRl IS43LL ISmR IS431mec ISSlS/T ISlF- AI ISi%-AI1 ISi%-AI11 IS-%R -

Fig. 2. Amino containing

acid sequence

identical

or (S, T) are underlined. above

the sequences,

C-terminal

alignment

for the putative

aa are boxed and conservative The two predicted as is numbering

and N-terminal

is a result of uncertainty

a-helix-turn-a-helix

of the alignment.

ends, respectively. in the nucleotide

transposases

replacements

nucleotide

sequence

Note that the IS43Zmec

The X occurring

sequence

of members

within the groups

of the IS257, recognition

and ISIS families.

motifs (Rouch

and ISIS-d111

in the IS431mec sequence

of DNA (Fig. 1).

ISSl

Positions

(D, E), (F, Y, W), (I, L, V, M), (H, K, R), (N, Q) sequences

at residue

et al., 1989) are shown are incomplete

11 (Barberis-Maino

at their

et al., 1987)

201

B. subtilis as the host compared

to E. coli; the oppo-

Thus,

only one such event

need

be proposed

to

site applies for ISIS-d1

isolated from Gram-negative

explain the available data; the last common

ancestral

bacteria.

are consistent

sequence for the three families of insertion

sequences

These results

that the IS257

and ISSl

families

with the idea have resided

in

Gram-positive hosts for some time and the IS15 family in Gram-negative hosts. This is not surprising, since transfer

of genes between

Gram-negative

bacteria

Gram-positive

and

is likely to be a rare event.

must have undergone such a transfer. The codon bias values for the IS elements preferred ISSlS,

host system,

of low to moderate

which is consistent genes

a

167 \

)_I?_ 161

ISS IS

b

1

fi

Time

Fig. 3. Evolutionary

IS15 n-1 ISIS-All Is15-AIII IS26, IS46,ISZ40

1SSl.S ISSlT

IS257R1, IS257L/R2 IS431L,IS431 R IS431mec

tree topologies

determined

for the three prototype

Di, of a particular

sequence

tree was calculated

sequences.

from its branch

from: Di = L { +

that includes

and reverted accounts

a Poisson

substitutions

for insertion

point in the unrooted + Xi, in

of substitutions

to the

correction

(Kimura

or deletion

for superimposed

and Ohta,

1972), and Xi

events, marked

by gaps in the

aligned

sequences

(Fig. 1). L is the effective

quence

in bp and Si is the raw substitution

length

contains

a different

the other two, u is the number contain entiating

unique nucleotides, positions

unique nucleotide differentiating relative IS257L

substitutions

positions

L = 842,

being

assuming prototype

evolutionary

uniform mutation sequences;

other

families are shown below.

set at equal to twice

d = 369,

e.g., for

u = 119, X, = 2, + 2 = 167.

tree derived from the unrooted rates, calculated sequenced

of

considered,

events for the sequence;

d, = 88,

the

to the number

Di = 842{ -314 ln(1 - 4/3[88 + 2 x 119(ss/~)]/842)} (b) The rooted

of differ-

dJZd distributes

sequence

X, was arbitrarily

to

where all sequences

in proportion

for the

of insertion/deletion

where

of positions compared

and zd is the total number

for the three sequences;

to all sequences.

the number

nucleotide

of positions

of the se-

score, determined

from: Si = d, + 2u(dJZd), in which d, is the number where the sequence

unrooted

The distance,

ln( 1 - 4/3 (S,/L))}

which the first term is the contribution distance,

with the parsi-

of Fitch (1971). (a) The only possible

tree,

for the three family

members

of the three

and to a

levels of expression,

with the proposed for transposase

(c) Selection for G + C content transposase proteins

ISIS-DI

/

tree structure

as encoding

correspond

nature of the polypeptides,

since such enzymes are usually produced amounts (e.g., Morisato et al., 1983).

IS257L

mony analysis

B. subtilis for IS257L

and E. coli for ISIS-d1,

prediction

in their

in low

and evolution

of

The G + C content of genomic DNA from different bacterial species varies between approx. 25% and 75% (Rosypal and Rosypalova, 1966). To allow encodement of proteins with similar activities across species, the diversity of G + C content in coding regions is accommodated for, in part, by the degeneracy in the genetic code, since synonymous codons vary in G + C content themselves (Bibb et al., 1984). Furthermore, the G + C content of the DNAs of each species is apparently under stabilizing selection, so that where a DNA molecule, which for example contains a resistance gene or transposon, is transferred from its original host to a new host with a different G + C content, the G + C content of the transferred DNA gradually alters to that of the new host (Sueoka, 1962). The question then arises of how the ability to encode for a function, such as a particular enzyme activity, is maintained under selection for an altered G + C content. The differing G + C contents of the related IS257L, ISSlS and ISIS-d1 transposase genes, which are 34.5%, 37.3% and 52.8%, respectively, permits examination of this question in terms of nucleotide and amino acid sequence changes. Nucleotide substitutions responsible for differences in G + C content between the reading frames of these transposase genes are distributed through most of the 205 codon positions examined for which the sequences had corresponding codons, excluding 15 invariant codon positions (Fig. 1). Among the codons for invariant amino acids, the majority of the mutation differences between IS257L or ISSI S and ISIS-d1 altered the G + C content, and were

202

naturally

restricted

positions. nucleotides codons, tution directly

in the most part to the third base

This indicates has occurred

that selection for particular at the third

and demonstrates theory

(e.g.,

applicable

position

1968;

in this situation.

amino acid composition of bulk cellular protein isolated from bacterial species with low or high

1983) is not

G t C contents (Sueoka, 1962) could have evolved, and also the evolution of amino acid composition

Conservative

biases in eukaryotes

that the neutral

Kimura,

in

genomes, the results explain how similar biases in the

substi-

with non-average

G

t C con-

subsets (D, E), (F, Y, W, (I, L, V, M), (H, K, R), (N, Q) or (S, T), involved G + C content changes at

tents, like Plasmodium falciparum (Hyde and Sims, 1987). It can also be inferred that transfer of a gene to a host with a different G + C content will acce-

all codon positions and most combinations of positions. These changes tended to result in an amino

maintenance

acid with A + T-rich codons

Thus,

amino acid replacements,

posases

encoded

that is exchange within the

occurring

by A t T-rich

DNA

in the transsequences

(IS257L and ISSIS) opposite an amino acid from the same subset encoded by G + C-richer codons in the ISIS-d1 sequence; for example, lysine (AAA) opposite arginine (CGG) at aa position 7(cf., Figs. 1 and 2) tyrosine (TAC) or phenylalanine (TTT) opposite tryptophan (TGG) at aa positions 20 and 129. In these cases, the lysine/arginine substitution can occur through three separate single nt substitutions, with the intermediate codons specifying lysine or arginine [i.e., AAA(lys)-AAG(lys)AGG(arg)-CGG(arg)], however, the aromatic amino acid replacements would require double substitution events to avoid encoding a non-aromatic residue in an intermediate step. At positions demonstrating non-conservative amino acid replacements there is a tendency for an amino acid with A t T-rich codons to occur in the A + T-rich sequences opposite any amino acid with G + C-rich codons in the G t C-rich sequences; for example, tyrosine (TAT) against proline (CCA) at aa position 3. In addition, in the ISIS-d1 sequence there are separate relative insertions of three arginine codons (CGG or CGC; aa positions 61,63 and 182) and two glycine codons (GGT or GGA; aa positions 120 and 189); and relative deletions of a tyrosine (TAT) and a phenylalanine (TTT) codon (aa positions 210 and 222) which positively contribute to the differences in G + C content. Taken together, these results account for the overall biases in amino acid compositions of the putative transposases, with a trend for more residues specified by A t T-rich codons, such as F, Y, I, M, N or K, to occur in the IS257 and ISSI sequences compared to the IS15 sequences, and vice versa for residues specified by G + C-rich codons, like P, R, A, and G. Furthermore, when extrapolated to whole

lerate polypeptide evolution

hastened

sequence

alterations

in a host with a similar G of particular

proteins

compared

to

t C content. would

be

by the shuffling of their genes between host

bacteria with differing G + C contents, as has probably occurred, for example, in the aminoglycoside phosphotransferase family (Thompson and Gray, 1983; Rouch et al., 1987); transfer may have aided in producing the wide variety of substrate profiles exhibited by the enzymes in this family, that is mirrored in the range of G t C contents (25-73%) among their encoding DNAs (D.A.R. and R.A.S., unpublished). Such transfer events will be rare given the likely impediments against transfer between unrelated species, such as restriction systems. However, this is not to deny their possible importance in the long term for the evolution of some genes. (d) Molecular organization and IS15 families

of IS in the IS257, ISSZ

Sequence conservation between members of the IS257, ISSl and IS15 families is reflected in their common molecular organization, as shown for their representatives in Fig. 1. The single significant open reading frame, encoding the putative transposase, accounts for approx. 85% of each IS element; the most highly conserved regions in the transposase polypeptide sequences specify two predicted cr-helixrecognition turn-a-helix nucleotide sequence domains (Fig. 2; Rouch et al., 1989), with a corresponding high degree of nucleotide conservation in their encoding DNA sequences (nt positions 169-229 and 302-361; Fig. 1). Also, although the lengths of the terminal IR vary between the IS257 (16-28 bp), ISSl (18 bp) and IS15 (14 bp) families (Rouch et al., 1989) there is good conservation of a 14-bp terminal IR with the left-hand IR containing the -35 box of the predicted promoter for the transposase gene of each sequence (Fig. 1). Since the

203

transposase

would

during transposition

be expected

to bind to the IR

(Craig and Kleckner,

may regulate its own expression

1987) it

as this would also

occlude the -35 promoter region from an RNA polymerase molecule. Although the -35 box is con-

impaired.

Also,

IS431 R has undergone

transition

at nt 2, which is in the terminal

outside end of IS431 R in Tn4004, of the IR is important

for transposition,

served, the -10 box and spacing between

these two

could render

Tn4004

and IS15

these results

suggest Tn4004

family members

is not;

IS257,

show spacings

ISSl

of 14, 16 and 20 bp,

respectively. The promoters are close enough to the ends of the IS elements that sequences flanking their left hand ends could affect expression, the case of IS257 with its sub-optimal spacing (Rouch et al., 1989). In addition,

especially

IR of the

so that if integrity as in other

cases (e.g., Huang et al., 1986), then this substitution

promoter

elements

a G -+ A

effectively

inactive.

Together,

is a defective

trans-

poson. (e) Conclusions

in

promoter expression

of the transposase may be influenced by promoters located outside an IS but able to direct transcription into its transposase gene; if above a certain strength, such externally directed transcription could outcompete the transcriptional block imposed by a transposase molecule bound at its -35 box. Hence, transposase gene expression, and therefore transposition activity, may be influenced by the site of insertion of these elements. In contrast, some other IS elements, such as IS5 (Kroger and Hobom, 1982) contain Rho-independent transcription terminators in their terminal regions which block outside to inside transcription. Partly due to lack of optional extras like these, insertion elements in the three IS families described here are among the smallest such sequences. Of these, the IS257 elements are the most compact, with minimal spacing between the potential promoter and ribosome-binding site (8 bp, cf. 23 and 22 bp for ISSl and ISIS families, respectively) and between the -10 and -35 regions of the promoter (14 bp cf. 16 and 20 bp for ISSI and IS15 families, respectively); and they also potentially encode for the shortest transposase, of 224 aa (cf. 226 aa for ISSl and 234 aa for ISIS). Interestingly, sequence differences between the IS257 sub-family and the two IS431 elements, which flank merA merB in the putative mercury-resistance transposon Tn4004 (Gillespie et al., 1987; Lyon and Skurray, 1987), suggest reasons for the failure to observe transposition of Tn4004 (Murphy, 1988); IS431 L has suffered a G --t A transition at a critical position in its ribosome-binding site (Fig. l), suggesting that expression of the transposase would be impaired, and if the transposase is preferentially cisacting, as occurs for a number of other transposable elements (McFall, 1986), transposition might also be

The degree of sequence similarity between members of the IS257, ISSl and IS15 families of IS leaves little doubt that they have a common ancestry. These IS elements, therefore, form a superfamily of IS, that has members prevalent among a number of Gram-positive and Gram-negative pathogens; in these organisms, it would seem that this IS superfamily has been associated with the spread of many antimicrobial resistance determinants in recent evolutionary times (Labigne-Roussel and Courvalin, 1983; Skurray et al., 1988; Rouch et al., 1989). The biases in amino acid substitutions in the transposase polypeptide, that correlate with the G + C contents of their encoding DNAs, suggest that the transfer of genes between hosts of different G + C contents will accelerate the evolution of their products, as a result of G + C content normalization. Similarly, divergence in G + C content of related species, whether prokaryotic or eukaryotic, would accelerate protein evolution on a genomic scale.

ACKNOWLEDGEMENTS

This work was supported by a Project Grant from the National Health and Medical Research Council (Australia).

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