FEMS Microbiology Letters 83 (1991) 11-16 © 1991 Federation of European Microbiological Societies 0378-1097/91/$03.50 Published by Elsevier ADONIS 037810979100403N

11

FEMSLE 04598

Identification of an insertion sequence, IS1081, in Mycobacterium boris D e s m o n d M. Collins and D i a n a M. S t e p h e n s Central Animal Health Laboratory, Wallaceville Animal Research Centre, Upper Hutt, New Zealand

Received 15 April 1991 Revision received 29 May 1991 Accepted 1l June 1991

Key words: Insertion sequence; Repetitive DNA; Tuberculosis; Mycobacterium bot,is; Mycobacterium tuberculosis

1. S U M M A R Y

2. I N T R O D U C T I O N

An insertion sequence, IS1081, in the genome of Mycobacterium boris has been identified and sequenced. It is 1324 bp long with 15 bp inverted repeat ends and contains a large ORF. There are six copies of IS1081 in the genome of M. boris and the element is also present in Mycobacterium tuberculosis. IS1081 is not closely related to other D N A elements described in actinomycetes but its putative transposase bears some resemblance to that of IS256 from Staphylococcus aureus. IS1081 may be useful for genetic manipulations and for developing a diagnostic test for bovine tuberculosis based on the polymerase chain reaction.

Tuberculosis in both humans and domestic animals is a worldwide problem on an enormous scale. M. tuberculosis, the major human pathogen is very closely related to M. boris the major veterinary pathogen [1]. Traditional diagnostic tests for these organisms have either been very slow or have lacked specificity or sensitivity [2]. The advent of the polymerase chain reaction, together with the discovery of repetitive D N A sequences that appear to be specific to M. tuberculosis and closely related species such as M. bouis, offers the promise of diagnostic tests that are fast, sensitive and specific [3-5]. However, the DNA elements identified in M. tuberculosis are not ideal for use in diagnostic tests for M. bovis. The best studied element is present in many copies in M. tuberculosis but is only present as a single copy in most M. boris isolates [3,6]. This limits the sensitivity of any test for M. boris based, on this element. Another repetitive ele-

Correspondence to: D.M. Collins, Central Animal Health Laboratory, Wallaceville Animal Research Centre, P.O. Box 40063, Upper Hutt, New Zealand.

12 E Sa

4.4 kb I I

1.95 kb

1.7 kb

Sa

E

I

[

Sa

Pv

X

PB

SH P

E

[

I

1

I I

II

I

Pv

X

PB

Sll P

E

I

I

[[

II I

[

I

pB1

pg3

pB5

Fig. 1. Restriction sites of inserts in pBl, pB3 and, pB5; B, BstEII; E, EcoRI; H, HindlII; P, PstI; Pv, Pt,uII; S, SacI: Sa, Sail: X, Xhol.

ment that has been identified in M. tuberculosis appears to have an imperfectly repeated sequence that is not an ideal template for the polymerase chain reaction [5]. In this present study we produced a partial genomic library of M. boris and screened it for repetitive elements. A similar approach was used to that which had previously been successful in identifying a repetitive element in Mycobacterium paratuberculosis [7].

3. M A T E R I A L S A N D M E T H O D S

3.1. Molecular cloning and screening Chromosomal D N A was extracted from M. boris T M C 410 (synonymous with neotype strain A T C C 19210) and digested with EcoRI. Fragments of size 1-7 kb were separated by electrophoresis, recovered using glass adsorption (Geneclean kit, Bio 101) and ligated into the E c o R I site of pBR328. Recombinant plasmids were cloned in Escherichia coli DH5 using standard techniques [8]. Recombinant colonies were cultured and then lysed on replica Nylon filters [7]. Genomic probes of M. boris T M C 410 and M. tuberculosis H37Rv T M C 102, labelled with 32p, w e r e hybridized to the filters for 20 h at 50°C in 50% formamide followed by a final stringent wash at 68°C in 0.1 x SSC, PIasmid D N A was prepared and restriction maps of plasmid inserts were obtained using standard techniques [8]. Subcloning of plasmid inserts was performed by digestion of plasmid D N A with the appropriate restriction enzymes, purification of the fragments

as above and ligation into pBR328 or M13mpl8 vectors. Strains of E. coil were then transformed with the constructs.

3.2. DNA sequencing and analysis The 'Erase-a-base' system ( P r o m e g a ) w a s used to make progressive, unidirectional, deletion mutants of two M13mp18 subclones which contained the repetitive sequence inserted in opposite orientations. Appropriately sized deletion mutants were cloned and chosen as directed by the manufacturers. Single stranded D N A sequencing of the inserts in these M13 mutants was performed by the dideoxy method using the 'Sequenase' kit (United States Biochemical Corporation) and standard methods [8]. The sequence was analysed using the package supplied by the University of Wisconsin Genetics Computer Group [9].

4. R E S U L T S A N D D I S C U S S I O N None of the 450 recombinant clones screened gave a more intense hybridization signal with the M. boris probe than with the M. tuberculosis probe. This indicated that none of the recombinant plasmids contained a D N A element unique to M. boris or a D N A element present in substantially more copies in M. boris than in M. tuberculosis. D N A from one clone hybridized more strongly to both probes than did D N A from other clones indicating that this clone contained a D N A element that was repeated a number of times in the genomes of both organisms. Investigation of this clone revealed that it contained a

13

plasmid (pB1) comprised of pBR328 and a 4.4-kb insert (Fig. 1). A probe made from pB1 hybridized to more than 12 fragment lines on Southern blots of DNA from ten strains of M. boL,is and two strains of M. tuberculosis that had been digested with BstEI1 or PstI. The insert in pB1 was digested with SalI and E c o R I and the resulting three fragments were subcloned using pBR328 or derivatives to produce plasmids of the expected size as follows: pB2, 0.1 kb; pB3, 1.95 kb (Fig. 1); pB4, 2.35 kb. Probes of these plasmids were hybridized to Southern blots of genomic DNA from M. boris that had been digested with Bst EII. The results (Fig. 2) clearly indicated that the repetitive element was present in pB3 and that pB2 and pB4 contained flanking sequences. Most of the insert in pB3 was subcloned into M13mp18 to form pB5 whose insert is shown in Fig. 1. A probe of pB5 hybridized strongly to ten fragment lines on a Southern blot of M. boris DNA that had been digested with BstEII (Fig. 2). This indicated that the repetitive element was a

b

c

d

e

f

kb 8.5--

4.3

m

Q h

2.3

i¢

-

m

m Q

U

m

m 1.4--'

0.7

m

o

~-

Fig. 2. Southern blots of DNA from M. bovis; digested with BstEII and probed with (a) pB1, (b) pB2, (c) pB4, (d) pB3, (e) pB5; (f) digested with Bcll and probed with pB5.

present in the pB5 insert and that part of a flanking sequence present in the insert of pB3 had been removed. Southern blots of M. boris DNA that had been digested separately with restriction enzymes BclI (Fig. 2), SmaI, PL,uII and B a m H I which do not have sites within the insert were exposed to a probe of pB5. In all cases the probe hybridized to six fragment lines indicating that there are six copies of the repetitive element in the M. bo~'is genome. The entire insert in pB3 was also inserted into M13mpl8 in the opposite orientation to the insert of pB5 to form pB6. All of the insert in pB5 and the corresponding part of the insert in pB6 were sequenced (Fig. 3). The 1712 bp segment contained an insertion-like element designated IS1081. This element is delineated by 15 bp inverted repeats between positions 333-347 and 1642-1656. IS1081 is 1324 bp long with a large O R F beginning at residue 385 in Fig. 3. This O R F occupies most of IS1081, is preceded by a ribosomal binding site, and codes for a putative protein of 415 amino acids. IS1081 also contains ten shorter ORFs some of which have possible ribosomal binding sites. A search of the EMBL and GenBank databases did not reveal any closely-related DNA sequences. The insertion sequences so far described in actinomycetes can be divided into two groups: those which do not have inverted repeats but are flanked by similar short sequences (ISl16 from Streptomyces clavuligerus and IS900 from Mycobacterium paratuberculosis [11]); and those with inverted repeats ( I S l l 0 and a mini-circle both from Streptomyces coelicolor [11] and IS986 [3] and the almost identical 156110 [4] from M. tuberculosis). IS1081 has characteristics of both groups as it contains inverted repeats but also has flanking sequences that show some similarity to those of IS900 and ISl16 (Fig. 4). Pairwise comparison of the large O R F putative proteins from all these insertion sequences to that of IS1081 showed that they had 20% or less identical residues. The putative protein product of the large O R F of IS1081 was screened against the N B R F and SwissProt databases. One sequence was identified with homology significantly above back-

14 GCCGAGCCGA

CAAGACATGC

CAGCGCAACC

CGCTTCATCG

TCGTGGCAGG

TGTTGGGCTG

70

ATTTTGGTCA

5 ' • . .G A A T T C G A T C ACCCAGCACC

TGCCAGGACG

GGCTACGGAT

GTACACGGCG

ACGACGGTAT

GGGAGGATGT

CCGGTCTTGC

150

TCCGGTCATG

TCCGGTGAAT

GTGCTGCCAA

CATCCTGGGG

ACCGTCCAGC

GAGTTTCACC

ACACCTTGGG

GCACCTTCTG

230

TCACTGCTCG

GTGCTGTGGA

TTGGTGTCAA

GTTACGTCCA

GGGGTGTGGT

GTACGGGCAG

GT~GGCCGG

TGGGCGTGTC

310

GTAGCCCAGT

AGTGGGCGGT

CATCGCGTGA

TCCTTCGAAA

CGACCAGCAA

~GTC~TCG

AAGGAAATGA

CGCAA~GA$C

390

C G A* G C A*G C T T*

C T*G G C*T G A C* C

~CTCG~AC

~ GG~GA~CC~G

GATCTGCTGC

GCG~GC~GC~

470

CTCGACGTTC ATCGCCGCCT . T G A T G G G G G C

TGA.AGCCGAC

G~CC~GT~CG

~GG~GG~CTA

CCGCG~C~C

A~CGATGAGC

550

GGTCCAATCA GCGCAACGGC TACCGCCACC

.GTGATTTCGA C A ~ C C ~ T G ~ C

G~CA~CG

ACGTCG~GA~

CC~C~GC~G

630

CGCCAGGGCA GCTATTTCCC G G A* C T G*G C T G*

C T*G C A*G C G *C C

~CAAGCGAG~

TG~C~CG~A

C~GA~CAGCG

TGG~GG~GA~

710

CTGCTACCTG CT.GGG.AGT.AT C C A C T C G . C C G

GATGGAGCGC

C~GGTCGAAA

~AC~TG~TG~

GA~AAAGC~T

T~C~GT~GC

790

.AAGT.G T C G A T

CAT. G G C C .AAA G A G C T C G A C G

.AAGCCGTAGA

GG~GT~TC~G

A~CC~CC~GC

TCGATG~CG~

CC~GTATA~C

870

TTCC.TCGCCG

CCGACGCCCT

*C A C A C C T T G A

T*C G*C C*A C*C G G

950

C G T.C A A.C G C C*

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

GGTGCTCAA.G G.TGCGCGAGG C* A G*G C C*G C G*T * C G*T C G* G A*G T G* GAGAGATCCT GGG.CATCCAG G ~ C A ~ C T ~ C G ~ C G A G G A C G ~

GG~CG~CT%G

C~GG~GT~CT

1030

T.CCGCGACCT

GGTCGCCCGC

GGCCTGTCCG GGGTCGCGCT * * * * * * * GG~CA~CA~C

GACG~CC~CG

~CG~CC~GG~

GG~CG~GA~C

Iii0

GGCGC.CACCC

TGCCCGCAGC

AG~C~TC~G

ATGG~AG~CA

~CC~G~GC~

1190

CTCCTGGCCG

TG.GGTGCGCA

GGCC~GC.AG CGC~GCAGAA ~ C C A C T A C G ~ CeCTGCTGCA e~CCA.~C~AC G A*C C A* G C*C C G*

*A G T* T G *T T G*C C * C ~ T A T G A T C

1270

GGGTACTCGA

CGCTCT.GACC

GACA~CG~CC

GCA~CGACC y GCTGG~GT~C

ACCGCCTTCC

CCAA. G C A G A T

GGAACGCCTC

AACCGAGAGG

TACGACGCCG

1430

A A C• C G A*C G T C.

G T* G G G.C A T C* T

GAC~,ACTCC e.CGCGGTGGC * CGAGCACC~C CZGGCGCC.AAATC~GG~CCA A C A A C C C C C A TccccG~ccG CGCC~rCGA.TC A ~ C C G C C ~ C G

~CGGAG~CG~

CC~CG~CGAA

C~CACGACG

1510

.AA~GGA.~CG÷ AGGAC.GGCGC TACC.TGGG.CC TCGAGGT.CC.T C A C C C G A G C C GCC~GCAGC .~CCACCA~ cAccccAGcA CTGACCACC~ A G A C T G C C A C

CGAGCAGCAC

TGACCAGCAC

CG~G~CCC

1590

CCGAAGGATC

ACGCGAGG~

CCTTCACTCG

1670

Tc~cTc.A~c ~A~CG~CAC

TACACCACGT

CCCTGGCCTT

GGCCTGGTGT

CAGGCCCAGC

A*C G C* C G *A A T*C

TG...3'

1350

1712

Fig. 3. Nucleic acid sequence of the insert of pB5. The 15 bp inverted repeats are underlined; *, codons of the large ORF.

g r o u n d , the putative transposase of IS256 from S. aureus [10]. T h e two putative p r o t e i n s share 35% identical residues a n d 54% residues c o n s i d e r e d to have acceptable conservative a m i n o acid changes. In S. aureus, IS256 is associated with a t r a n s p o s o n c o n t a i n i n g antibiotic resistance genes. W h e t h e r IS1081 has a similar association r e m a i n s to be d e t e r m i n e d . However, IS1081 may be useful for genetic m a n i p u l a t i o n of the g e n o m e s of both M. boris a n d M. tuberculosis. T h e suitability of the s e q u e n c e of IS1081 for use in a diagnostic test for tuberculosis organisms b a s e d o n the polym e r a s e chain reaction is yet to be investigated. I S 900

TGGTCATGTGGTGT

.... CTCCTTCGCG

I S 116

TCGGTCATGGTCGG

.... TCTCCTGGTG

IS 1081

GTAGTGGGCGGTCA

.... GGAACCTTCA

Fig. 4. Proposed insertion sites of three actinomycete elements. Nucleotide sequences shared by all three sites are underlined.

ACKNOWLEDGEMENT This work was s u p p o r t e d by the New Z e a l a n d A n i m a l H e a l t h Board. W e t h a n k G.W. de Lisle for his e n c o u r a g e m e n t a n d helpful advice.

REFERENCES

[1] Wayne, L.G. (1984) ln: The Mycobacteria: a Sourcebook (Kubica, G.P. and Wayne, G, Eds.), pp. 25-65. Marcel Dekker, NY. [2] Pao, C.C., Yen, T.S.B., You, J., Maa, J., Fiss, E.H. and Chang, C. (1990) J. Clin. Microbiol. 28, 1877-1880. [3] Hermans, P.W.M., van Soolingen, H.D., Dale, J.W., Schuitema, A.R.J., McAdam, R.A., Catty, D. and van Embden, J.D.A. (1990) J. Clin. Microbiol. 28, 2051-2058. [4] Eisenach, K.D., Cave, M.D., Bates, J.H. and Crawford, J.T. (1990) J. Infect. Dis. 161,977-981. [5] Patel, R.J., Fries, J.W.U., Piessens, W.F. and Wirth, D.F. (1990) J. Clin. Microbiol. 28, 513-518. [6] Thierry, D., Brisson-Noel, A., Vincent-Levy-Frebault, V., Nguyen, S., Guesdon, J. and Gicquel, B. (1990) J. Clin. Microbiol. 28, 2668-2673.

15 [7] Collins, D.M., Gabric, D.M. and de Lisle, G.W. (1989) FEMS Microbiol. Lett. 60, 175-178. [8] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory', Cold Spring Harbor, NY. [9] Devereux, J., Haeberli, P. and Smithies, O. (1984) Nucleic Acids Res. 12, 387-395.

[10] Byrne, M.E., Rouch, D.A. and Skurray, R.A. (1989) Gene 81,361-367. [11] Leskiw, B.K., Mevarech, M., Barritt, L.S., Jensen, S.E., Henderson, D.J., Hopwood, D.A., Bruton, C.J. and Chater, K.F. (1990) J. Gen. Microbiol. 136, 1251-1258.