Reversal of Mu gem2ts-induced mutations

ELSEVIER

FEMS Microbiology Reviews 17 (1995) 171-176

Reversal of Mu

MICROBIOLOGY REVIEWS

gem2ts-induced mutations

P. Ghelardini ~'*, J.C. Li6bart b G. Fabozzi a,~, B. Tomassini c, R. D ' A r i b9 L. Paolozzi

c

a Centro di Studio per gH Acidi Nucleici de[ CNR, e / o Dipartimento di Genetiea e Biologia Molecolare, Ifniversit~ di Roma 'La Sapienza" Piazzale A. Moro 5, 00185 Rome, Italy b institut Jacques Monod, CNRS, Universit~ Paris 7, Paris, France c Dipartimento di Biologia, Universith "Tor Vergata ; Rome, Italy

Abstract Mutations induced by the integration of a Mu gem2ts mutant prophage can revert at frequencies around 1 × 10-6, more than 104-fold higher than that obtained with Mu wild-type. Several aspects characterize Mu gem2ts precise excision: (i) the phage transposase is not involved; (ii) the ReeA protein is not necessary; and (iii) revertants remain lysogenic with the prophage inserted elsewhere in the host genome. In addition, prophage re-integration seems to be non-randomly distributed, whereas Mu insertion into the host genome is a transposition event without any sequence specificity. In this paper, we describe that the site o f re-integration somehow depends on the original site o f insertion. Two alternative models are proposed to explain the strong correlation between donor and receptor sites. Keywords: Bacteriophage Mu; Precise excision; Transposition

1. Introduction

Mu is a genetic element which developed a double way of life, as temperate bacteriophage and as transposable clement. As a prophage, Mu DNA is passively replicated with the bacterial chromosome, whereas, after induction of a lysogen, the lytic cycle is started and new copies of Mu D N A are formed by random replicative transposition throughout the bacterial genome, causing various types of rearrangements, such as deletions, inv~,~::~ions and replicon fusions [1,2]. During this process, Mu DNA never excises from its original target site and free phage DNA is never found in the cytoplasm (for review, * Corresponding author. Tel.: + 39 (6) 445 3320; Fax: + 39 (6) 4991 2500

see [3]). At the end of the lytic cycle, each molecule of virion DNA is encapsulated and liberated from the host cell, conferring infectivity to t h e transposon. Nevertheless, in spite of its behaviour as a transposable element, mutations due to the integration of Mu DNA are remarkably stable: reversion frequencies a r e less than 10 -1° [4]. This lack of reversion of Mu-induced mutations is paradoxical when compared with the behaviour of other transposable elements (both prokaryotic and eukaryotic), all of which are able to excise precisely. The first observations on Mu DNA excision were performed by Bukhari [5] with bacterial strains lysogenie for a phage mutant (Mu cts62 dX) able to express the transposase after induction of the lyric cycle by thermal shift, without either host cell killing or phage particle production. Under these conditions,

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P. Ghelardini et aL / FEMS Microbiology Reviews 17 (1995) 171-176

both precise and imprecise excision events were obtained, all of which associated with prophage loss. Mu cts62 dX excision required partial derepression of the Mu A gene, coding for the transposase, and was RecA-dependent [6], unlike other prokaryotic transposons. For example, TnlO excision is independent of RecA [7,8] and of transposase [7]. Analogously, Tn5 excision is similar to spontaneous deletion events [9] without any involvement of the transposase. The excision properties of Mu gem2ts prophage have been described recently [113]. The gem2ts mutation of a Mu prophage induces profound phenotypic effects on the bacterial host which can be observed in the presence of viral immunity [11]. In particular, Mu gem2ts prophages alter the DNA superhelical density and affect expression of a large number of host genes. Considerable evidence suggests that the

A 1

primary target of Mu gem2ts action is the activity of the B subunit of DNA gyrase [12,13].

2. Properties of Mu gem2ts excision From Escherichia coli lysogens in which Mu gem2ts DNA was integrated in the malT, thyA or lacZ gene, Mal +, ThyA + and LacZ + revertants were readily obtained at frequencies around 1 × 10 -6. These reversion frequencies are more than 104-fold higher than those obtained with Mu gem +.

2.1. The excision of Mu gem2ts is precise A careful study on the precision of the excision events giving rise to revertant clones was carried out with lysogens with the Mu gem2ts prophage inserted

B 2

3

4

5 > 9 kb

Bacterial Strain RS54 R354 lauZ::Mu ~ m ~ t s

Miller Units of 8-galactosidase 1569 I

R364 Laa+ (Mu ~m2ts)

1373

R365 Lac+ (Mu ga~.ts)

1627

R366 Dao + (Mu ger~ts)

1568

.., 1891 bp .., 1 5 3 2 bp Fig. 1. (A) Restriction analysis of the lac region of Lae ÷ revertants. DNA of the non-lysogenic strain RS54, lac::Mu gem2ts lysogen R354, and Lac ÷ revertants R364, R365 and R366 was extracted and digested with restriction enzymes EcoRI and EcoRV. After agarose gel eleetrophoresis, the gel was blotted onto an Amersham hybrid N membrane and then hybridized as described by Maniatis et al. [15], using as a probe plasmid pMC1403, which carries the E. coli facZ lacy genes. Lane 1, RS54; lane 2, R354; lane 3, R364; lane 4, R365; lane 5, R366. (B) ~-Galaetosidase activity produced by the Lac + revertants. The test was performed as described by Miller [16].

P. Ghelardini et al. / FEMS Microblotogy Reviews 17 (1995) 171-176 A

B

m

¸

~,-¸

A V M U left e n d G A C G C GCGIUlITGflflI"fATGGC C CIICflCCAGTGGCGCG GCGACTGTIZITGIII"TCIZCTTGA flGIAC

B

GnCGCGCGAAITGAnlTnTG(;C~{:ACACCA(;T6GCGCSGCI;Rcrrcc nGTrc~IRCaTCAGc¢~CT Fig. 2. Sequence of lacZ gene of lacZ::Mu gem2ts lysogen R354 and of Lac ÷ revertant strain R364. (A) The precise point o f insertion of the prophage in the IacZ gene in strain R354 was localized as described in [10]. The fragment containing the lacZMu gem2ts junction w a s then sequenced by linear PCR using an o]igonucleotide from lacZ proximal to the junction point (5'TGACCGCTGGGATCTGCCAT-3') as primer. (B) A fragment containing the iacZ gene from revertant strain R364 was amplified by PCR and sequenced as above. The sequence obtained was then compared with the lacZ sequence o f GenBank, accession number J01636.

in the lacZ g e n e . As a first step, we performed a Southern blot analysis of the lac region of three independent Lac + revertants, together with the lysogenie and the non-lysogenic parent. The results showed recovery of the wild-type digestion pattern in the Lac + revertants (Fig. 1A). The functionality of the lac operon, after excision of Mu DNA; was deduced by the normal production of fl-galactosidase activity in the three revertants: about 1500 Miller units. This value is similar to that obtained with the parental non-lysogenic Lac÷ strain, whereas the lacZ::Mu gem2ts parental strain produced only 1 Miller unit of fl-galactosidase (Fig. 1B). Finally, sequence analysis showed that the wild-type lacZ sequence had been restored in the revertants (Fig. 2).

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2.2. The excised prophage is non-randomly re-integrated The reversion rates described for Mu gem2ts are comparable to the excision frequency of Mu cts62 dX [5]. There are several significant differences, however, between Mu gem2ts and Mu cts62 dX. First of all, revertants obtained by excision of Mu cts62 have no trace of Mu DNA, whereas those obtained by excision of Mu gem2ts remain lysogenie. Second, the X mutations studied by Bukhari are absolute lethals for Mu development, so excision is observed under conditions in which lytie growth is not possible; however, with Mu gem2ts, which is a temperature conditional lethal for plaque formation, excision is observed at permissive as well as nonpermissive temperatures. In addition, Mu cts6z d X excision is RecA-dependent, whereas Mu gem2ts excision is not. Finally, excision of Mu cts62 dX is observed only at or above 37°C, when there is partial derepression of the Mu transposase (gene A product), whereas excision of Mu gem2ts does not require a functional transposase: it is observed in cultures of fully immune (c +) lysogens and with a prophage carrying an unsuppressed Aam1093 mutation. The excision of a Mu gem2ts prophage from the malT, lac and thyA genes is apparently always accompanied by re-integration, generally giving a monolysogen. For the Mu gem2ts prophage excised from malT, the 94-rain region of the E. coli chromosome seems to he a preferential re-integration site [10] . . . . . In order to generalise these observations and in particular to determine whether or n o t 94 min is a specific site for re'integration, we extended our analysis to other lysogens in which the prophage was integrated in different genes (Table 2 ) . The results shown in Table 1 indicate that it is also possible to observe reversion for mutations due to Mu gem2ts insertion in the gai, man and xyl genes. As before, Table 1 Reversion o f Mu gem2ts-indnced mutations Strain

Selection

Frequency

R370 gal::Mu gem2ts R371 man::Mu gera2ts R372 xyl::Mu gem2ts

Gal + Man + Xyl +

7.0× 10 - 7 7.4 × 1 0 - 7 1.1×10 - 7

P, Ghelardini et aL / FEMS Microbiology Reviews 17 (1995) 171-176

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the revertants obtained remain lysogenic, with a Mu

gem2ts prophage inserted elsewhere in the host

A

B 1 23456

genome.

7

1234567

3. Localization of the insertions obtained after excision o f M u gem2ts For a first localization of the prophage present in the revertants of Mu gem2ts lysogens, we used pulsed-field agarose gel electrophoresis on chromosomal D N A digested with the restriction endonuclease NotI. The digestion patterns of the lacZ::Mu gem2ts lysogens R353 and R354 showed a new band around 400 kb (Fig. 3A, lanes 3 and 4). This was expected, since the lacZ gone lies in a 360-kb NotI fragment, the migration of which will be increased to 400 kb b y the insertion of the 37-kb of the Mu genome. The digestion patterns of the non-lysogenic parent strain RS54 and the Lac + revertants (namely R365 and R366, derived from R354 and R368 and R369 derived from R353) were indistinguishable. The gel was then blotted onto a membrane and hybridized with the internal EcoRI-EcoRI fragment of the Mu genome (Fig. 3B). The non-lysogenie strain RS54 gave no hybridization signal, whereas the lysogens R354 and R355 gave a signal in the 400-kb band, as expected from the prophage localization in lacZ. The four revertants showed hybridization in the 1000-kb band. We next mapped the re-integrated prophages by conjugation and P1 vir-mediated transduetion. In all cases, the prophage was localized in the malt gene, which indeed lies in the 1000-kb NotI fragment [14]. The location of the prophage in Gal ÷ revertants was determined analogously by Southern analysis of Table 2 Bacterial strains Number

Genotype

Origin

RS54 R353 R354 R362-366 R368-369 R370 R371 R372 R373-374

relAl araD su ° RS54 lacZ::Mu gem2ts RS54 lacZ::Mu gem2ts RS54 LacZ + (Mu gera2ts) RS54 LacZ + (Mu gem2ts) RS54 gal::Ma gem2ts RS54 man::Mu gem2ts RS54 xyl::Mu gem2ts RS54 Gai + (Mu gem2ts)

Laboratory collection Ghelardini et al. [10] Ghelardini et al. [10] Lac" revertants of R354 Lac + revertants of R353 This work This work This work Gal + revertants o f R370

Fig. 3. (A) Pulsed-field gel electrophoresis of Lac + revertants. D N A of non-lysogenie strain R554 (lane 7), lacZ::Mu gem2ts lysogens R354 and R353 (lanes 3, 4), Lac + revertants R365-366 (lanes 1, 2) and R368-369 (lanes 5, 6) was extracted, digested with NotI and analysed as described in [10]. Arrowheads indicate migration o f yeast DNA pulsed-field gel electrophoresis markers (Pharmacia): 555, 450, 375, 295 and 225 kb. (B) Localization of the Mu prophage in Lae ÷ revertants. The gel shown in Fig. 2A was blotted onto an Amersham hybrid N membrane and then hybridized as described by Maniatis ct al. [15], using as a probe the internal EcoRI-EcoRI fragment of Mu DNA. Arrowheads indicate the position of 1000-and 400-kb fragments.

the chromosomal D N A extracted from the gal::Mu gem2ts lysogens and from revertant clones digested with Notl (Fig. 4). In this case the prophage moves from the 17 min position of the gal operon (a 206-kb fragment generated by NotI digestion) to a region between 2 and 5 rain on the E. coli map, in the 270-kb NotI fragment.

4. Conclusions We previously reported that Mal + revertants obtained after excision of Mu gem2ts from the malT gone are still lysogenic with the prophage inserted in a site at 94 rain on the E. coli chromosome. However, from malT::Mu gem2ts lysogens, targeted reintegration could also be obtained, for example in the thyA gene, although at a lower frequency [10]. This

P, Ghelardini et al. /FF, MS Microbiology Revlews 17 (1995) 171-176 kb

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350

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300

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250

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150

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Fig. 4. Localization of the Mu prophage in Gal + revertants. DNA of gah:Mu gem2ts iysogeu (lane 1) and Gal + revertants R370371 (lanes 2, 3) was extracted, digested with NotI and hybridized as described in the legend to Fig. 2. Arrowheads indicate the position of the 240-kb (corresponding to the insertion of 37 kb of Mu DNA into the 206-kb fragment containing the gal gene) and 310-kb fragment {270 kb plus Mu DNA).

non-random re-integration is a characteristic of the excision-re-integration process of Mu gem2ts, since the Mu DNA insertion into the host genome is generally a transposition event without sequence specificity. To understand how the 94 min site is specific for Mu gem2ts integration after excision from another genetic locus, we have studied the re-integrations obtained after excision from two loci: lacZ and gal. In both cases, we obtained re-integration at specific sites different from each other and from the site at 94 rain, suggesting an aimed re-integration depending on the original ('donor') site of the insertion. At present, to explain the strong correlation between donor and receptor site of Mu gem2ts, two models can be proposed. The first postulates the existence of microhomologies and the second o f some topological constraint between the two sites involved in phage DNA migration. Although the first hypothesis cannot be formally eliminated, it is made unlikely by the fact that the prophage excised from various sites of the malT gene always shows preferentiality for the 94 rain site [10]. The topological

175

model on the other hand postulates that the distance between donor and receptor sites is determinant, which, although genetically far apart, could be brought closer by nucleoid folding. The proximity between donor and receptor sites allows the illegitimate recombination event, i f the latter hypothesis is true, these observations could allow an approach to the study of nueleoid organization. Other experiments will be necessary to clarify these interesting questions.

Acknowledgements This work was partially supported by the "Fondazione Cenci-Bolognetti Istituto Pasteur" and "Progetto Bilaterale di Ricerca" from the CNR (Italy), the Ministero dell'Universith e della Rieerca Scientifica e Tecnologica (Italy) and the Association pour la Recherche sur le Cancer (France).

References [1] Faelen, M. and Toussaint, A. (1980) Inversions induced by bacteriophage Mu-1 in the chromosome of E. coli K12. 3. Baeteriol 142, 391-399. [2] Desmet, L., Faelen, M., Lef'ebvre N., Resibois, A., Toussaint, A. and van Gijsegem, F. (1981) Genetic study of Mu mediated chromosomal rearrangements. Cold Spring Harbor Syrup. Quant. Biol. 45, 355-363. [3] Harshey, R.M. (1987) Integration of infecting Mu DNA. In: Phage Mu (Symonds, Tonssaint, van de Putte and Howe, Eds.), pp. 111-136. Cold Spring Harbor L~boratory, Cold Spring Harbor, NY. [4] Jordan, E., Saedler, H. and Starlinger, P. (1968) O° and strong-polar mutations in the gal operon are insertions. Mol. Gen. Genet. 102, 353-363. [5] Bukhari, A.I. (1975) Reversal of mutator phage Mu integration. J. Mol. Biol. 96, 87-99. [6] Khatoon, H., Chaeonas, G., DuBow, M. and Bukhari, A.I. (1979) The Mu paradox: excision versus replication. In: Extraehromosomal DNA (Cummings, Borst, Dawid, Weissman and Fox, Eds.), pp. 143-154. ICN-UCLA Symposia on Molecular and Cellular Biology, Vol. X-V. Academic P~ess, New York, NY. [7] Gavron-Burke, C. and Clewell, D.B. (1982) A tmnsposon in Streptococcus faecalis with fertility properties. Nature (Lond.) 300, 281-284. [8] Lyon, B.R. and Skurray, R. (1987) Antimicrobial resistance of Staphylococcus aureus: ge.netic bagis. ,M,,.,,,b Rev. 51, 88-134.