Molecular Microbiology (1991) 5(9), 2165-2170

Characterization of two insertion sequences, IS701 and\S702, from the cyanobacterium Ca/of/7r/x species PCC7601

D. Mazel,^ C. Bernard,* R. Schwarz,^ A. M. Castets, J.Houmard, and N. Tandeau de Marsac*Unite de Physiologie Microbienne (CNRS, URA1129),Departement de Biochimie et Genetique f^oleculaire,Institut Pasteur, 28 rue du Dr Roux. 75724 Paris Cedex15, France.

Summary

We describe the characterization of two insertionelements, \S701 and \S702, isolated from Calotbrixspecies PCC 7601. These insertion elements werecloned from spontaneous pigmentation mutants. Bothshow the characteristics of typical bacterial insertionsequences, i.e. they present long terminal invertedrepeats and they duplicate target DNA upon insertion.These elements share no homology with the only othercyanobacterial insertion sequence described so far,\S891. At least 15 copies of IS 701 and 9 copies of IS 702were detected by hybridization experiments in theCalothrix 7601 genome. Their occurrence in severalcyanobacterial strains is also reported.

In^oduction

Cyanobacteria are the only prokaryotes that share withhigher plants and eukaryotic algae the ability to performoxygenic photosynthesis. To harvest light energy cyano-bacteria have developed antenna-pigment complexes,called phycobilisomes, which consist of brightly colouredphyoobiliproteins associated with a few non-chromopho-rlc linker polypeptides (Glazer, 1989). Because of theabundance of the phycobtliproteins in the cells (up to 50%of the total cell protein), pigmentation mutants are easilydetected on plates. Some years ago, the frequent appear-ance of spontaneous pigmentation mutants in cultures ofCalothrix 7601 (about 10"*), as well as the variabilityobserved in the plasmid patterns found in various subcul-

Received 26 July. 1990; revised 4 June. 1991. Present addresses; tUnit6de Physiologie Celluiaire, Departemenl des Biotechnologies, InstilutPasteur. 25 rue du Dr Roux, 75724 Paris Cedex 15, France: JLaboratoiredes Biomembranes et Surfaces Cellulaires (CNRS URA311), EcoleNormale Superieure, 46 rue d'Ulm, 75005, Paris. France: §Department olBotany, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel.•For correspondence. Tel. (1) 45 68 84 15: Fax (1) 43 06 98 35.

tures of the same strain, led to a belief in the existence ofmobile genetic elements (Tandeau de Marsac, 1983).

Until now, besides the DNA rearrangements that occurduring heterocyst differentiation (Golden ef a/., 1985;Golden, 1988) and the discovery of families of highlyrepeated short sequences, the STRR sequences, wide-spread in the genome of heterocystous strains (Mulliganand Haselkorn. 1989; Mazel etai, 1990), little is knownabout the dynamic organization of the cyanobacterialgenome. The only cyanobacterial insertion sequence (IS)characterized so far (ISS97 in Anabaena sp. strain M-131)has novel properties (Bancroft and Wotk, 1989) in that itlacks the typical terminal inverted repeats (IRs) and doesnot duplicate its target DNA sequence upon insertion. Afamily of putative ISs with a typical bacterial structure hasalso been found in the DNA oi Anabaena 7120, but theirtransposition ability has not yet been demonstrated (J.Alam and S. E. Curtis, cited In Bancroft and Woik, 1989).Recently, Cai and Wolk (1990) have reported the isolationof at least one other mobile DNA element in Anabaena7120 (hereafter designated Nostoc 7120 according to R.Rippka, personal communication, and Damerval et al..1989).

The phycobilisomes of Calothrix 7601 are mainly coni-posed of three phycobiliprotein classes: (i) allophyco-cyanin (AP), (ii) phycocyanin (PC) and (iii) phycoerythrin(PE). All the genes encoding the subunits of these differentphycobiliproteins have been characterized and the con-ditions for their expression are now well documented (forreviews, see Tandeau de Marsac ef ai. 1988; 1990).Comparison of the nucleotide sequences of the genes thatencode the three different Calothrix 7601 phycocyanins(PCI, PC2 and PC3) demonstrate that they share a highdegree of identity (approximately 74% for the cpcB genes(B^^ subunits) and 80% for the cpcA genes (a*'^ subunits)(Capuano ef ai. 1988; Conley et al., 1988; Mazel et al..1988). Consequently, cross-hybridization could be expec-ted in experiments performed by using a probe corres-ponding to one of these genes.

In order to isolate the suspected mobile elements ofCalothrix 7601. we anaiysed a selection of spontaneousmutants that showed an alteration in the PE:PC ratio. Inthe course of these studies, we detected three differentISs in the Calothrix 7601 genome, namely IS70^ \S702

2166 D. Mazefetai

GY3: \S701

PROBEBg Bg

BR1: IS 702

cpcH2 epeiz

500 Dp

Fig. 1. Localization and relative orientation of IS70f in mutant GY3 and of IS702 in mutant BR1 with respect to the cpcr and cpc2 operons, respectively.Open boxes indicate the structural genes. Boxes atjove the genes indicate the location of the ISs; ORFs are indicated by the an-ows. The hatched ends ofthe ISs represent the IRs. Bg, Bglil. R, EcoRI; Xb. Xba\.

and \S703. and characterized the first two. We report heretheir properties, together with the results of a screening fortheir occurrence in the genomio DNA from severaldifferent cyanobacterial strains.

insertion had occurred within the orfX gene, in an Xba\fragment (691 bp long in the WT genome), iocatedupstream from the cpc2 operon. The physical maps ofthese two Eco RI DNA fragments are presented in Fig. 1.

Results and Discussion

Cloning of insertion elements

A 1 kb H/ndlil DNA fragment covering opcBI and the 5'end of cpcAl (shown in Fig. 1) was hybridized to thegenomic DNA extracted from 15 mutants. Four of ttiem,nameiy GY1. GY3, GY4 and BR1, showed an alteration ofthe Eco Ri restriction pattern that corresponded to the cpcgenes. The EcoRI fragment carrying the cpcB2A2H2l2D2operon was 6.5 kb long in the wild-type (WTl strain (Mazeiet al.. 1988), whereas it was 7.6kb iong in the mutant BR1(Fig. 2). The three other mutants showed a difference in thesize of the EooH\ fragment bearing the cpcSMtEoperonwhich, in the WT strain, was 4.5 kb long (Mazel etai, 1988).In the GY3 and GY4 mutants, the size of that EcoRIfragment increased to 5.9 kb, while in GY1 it decreased to3.5 kb (Fig. 2 and data not shown). Further hybridizationexperiments, coupled with restriction analysis, demon-strated that the DNA rearrangements resulted from theinsertion of a piece of DNA for BR1, GY1 and GY3, andfrom a 1 kb deletion for GY4 (data not shown).

The 7.6kb EcoRI fragment from BR1 and the 5.9kbEcoRI fragment from GY3 were isolated from partiallibraries constructed in pTZ18R. In mutant GY3. theinsertion had occurred inside the cpcF gene, in a BglWfragment (478 bp long in the WT genome), locateddownstream from the cpc7 operon. tn the BR1 mutant, ttie

Characteristics of IS70^ and IS702

The GY3 BglW fragment described above was sequencedon both strands and its nucleotide sequence compared

GY3 WT BR1

kb

9,5

7,6

6,5

5,9

4,5

Fig. 2. Auforadiogram of Southern blots of the Calothrix 7601 GY3mutant {lane GY3), the wild-type strain (lane WT) and the BR1 mutant(lane BR1). Total DNAs digested with EcoRI were probed with a 1 kbH/ndlll fragment covering cpcBI and the 5' end of cpcAI. The sizes ofthe different hybridizing fragments are indicated on the left.

Cyanobacterial insertion sequences 2167

with the corresponding WT sequence. This comparisonprecisely located the insertion sequence 46bp down-stream of the BglW site. Sequence analysis revealedthat the inserted DNA presented a typical bacterial ISstructure surrounded by the direct duplication of a 4 bp WTsequence (TTAG). This IS, named \S701. is 1389bp longand is bounded by two imperfect IRs that are 25 bp longand differ in three positions. \S701 shows no homoiogywith any of the ISs previously described and, in particular,with the only well-characterized cyanobacterial IS, \S891.Translation of both strands of the \S701 sequence in thethree reading frames revealed only one open readingframe {ORF) of more than 100 codons. This ORF couldencode a 380-amino-acid polypeptide (Fig. 3). This puta-tive transposase shows only slight homology (approxi-mately 25%) with the C-terminal extremities of thetransposases from \S231, \S50R. IS4, \S10R, \SSH1 and\S186. This homoiogy corresponds to that previouslydescribed between bacterial transposases (Mahillon etai,1985), the five residues underiined in Fig. 3 being identicalin all seven sequences.

The nucleotide sequence of the Xba I fragment isolatedfrom the BR1 mutant showed that the inserted sequencewas precisely located 476bp downstream from the Xba\site, it also showed a typical bacterial IS structure, theduplicated WT DNA target for this insertion being 3 bp long(CGT). This IS, named \S702. is 1098bp long and pos-sesses two IRs of 40 bp which differ in seven positions.Translation of its sequence showed only one significantORF. If one considers the first ATG codon, the putativeencoded transposase is 260 amino acids long, but in thesame reading frame, 16 codons upstream of this codon,there is a GTG codon preceded by a potential ribosome-binding site which could give a product of 276 residues forthat ORF (Fig. 3). The encoded protein, whatever itslength, shows homology neither to the sequences ofknown transposases nor to any of the sequences of thePseqIP data base (Claverie and Bricault, 1986). Neverthe-less, as for the putative transposase encoded by the \S891from Anabaena sp. strain M-131 (Bancroft and Wolk,1989), its highly basic composition (22.4% Lys+Arg. andonly 9% Asp+Glu) is consistent with the DNA-bindingproperty required for transposases.

IS707 and IS702 share a structural particularity. In the IRsequence located upstream from their putative trans-posase, both ISs contain a sequence very homologous tothe -35 OTGACA) and -10 (TATAAT) consensussequences of the canonical Escherichia co//promoter (Fig.3). In fact, the identity between the two IS sequences inthis region is larger. They share a quasi-identical stretch oft i b p (around the -10 sequence), located in the samerespective position in both ISs (Fig. 3). The role of thesesequences is still unknown, but they could be implicated inthe regulation of transposase expression and reflect a

common mechanism for the control of transposition of\S701 and \S702.

In insertion events, the specificity for a particularsequence is variable among the different ISs and even thelength of the sequence that is duplicated can varybetween two insertion events for a given IS (Galas andChandler, 1989). The analysis of other insertion eventsimplicating IS70T and \S702 would be necessary in orderto establish to what extent the duplicated targetsequences, TTAG for IS70r and CGT for \S702, as well astheir lengths, are specific.

Frequency and distribution of /S701 and IS702 amongvarious cyanobacteriai strains

Hybridization experiments between probes, specific foreither IS7O7 or \S702, and total genomic DNA fromCalothrix 7601 were performed (Figs 4A and 4B). At astringency allowing up to 35% mismatching, the \S701probe revealed at least 15 different H/ndlll fragments (Fig.4A), while the \S702 probe revealed at least nine differentH/ndlll fragments (Fig. 4B). In orderto estimate if these ISsoccurred in other oyanobacterial strains, the same probeswere hybridized to total genomic DNA extracted fromvarious strains {Figs 4A and 4B, and data not shown). No\S701-\\ke sequence was detected in Nostoc 7120 and8009, Synechococcus 6301 or Synechocystis 6803, andno \S702-\\ke sequence was observed in Pseudanabaena6406, 6802, 6901, 7402, 7403, 7409, 7429 and 7367,Nostoc 6411, 6705, 6719, 7118, 7119. 7120 and 7413,Synechococcus 6911 or Oscillatorta 6412 and 6506. Withthe exception of Microcystis 7813 and 7820, both of whichgave an identical hybridization pattern (Fig. 4B), strain7813 being in fact a mutant of strain 7820 deficient ingas-vesicle formation {Damerval et ai, 1989), in all theother strains examined {Caiothrix 7504 and 7601, Pseuda-nabaena 6903 and 7955, Cyllndrospermum 73101, Nos-toc 6720, 8009 and 73102 or f\Aicrocystis 7806 and 7820)the distribution of the two ISs varies even between strainsbelonging to the same genus {Figs 4A and 4B). Inparticular, there are at least 15 copies of IS 707 in Caiothrix7601 but only two in Caiothrix 7504, two strains which aretaxonomically very closely related {Lachance. 1981:Damerval etai., 1989; Mazel etai., 1990). Since the IS copynumber varies even within a given genus, it cannot beconsidered as a useful taxonomic criterion. Similarvariations in the copy numbers have been describedalready for several ISs from E coli {Galas and Chandler,1989).

The iS702 probe cross-hybridized with DNAs fromnumerous strains that belong to different cyanobacterialgroups, i.e. unicellular (Microcystis PCC 7806 and 7820);filamentous non-heterocystous (Pseudanabaena 6903and 7955), as well as filamentous heterocystous strains

2168 a Maze/etal.

\S701

N Y K S L F P D V B S F E A F K Y L H V G C I S D L K R K T L P E I A K I V G L

140 160 l«0 200 220 240

D N Q 0 C L H H F L T T 5 P M D I E K L R T L R I . E L I L Q V L K G R P I I L ISATIiACCWKfcAGGGTTGCMCMTTTCTAACTACMCACCTTGGGATXTACWJJVGTTXAG>ACCTTIiAGGTTfcG».GTTIATTTTIi.CAAGTGCTAAAAGGTAGACCAA.TCfcTTTT*ATT

2«0 2*0 300 320 340 3C0

I D E T G D K K K G S K T D Y V K H Q Y l G N L G K T D N G I V A V T V Y G V rUTTGATGAGACAGGGGATAAAAAGAAACGGAGCAAGACAGATTATGTGAAACGGCAGTATATAGGAAATTTGGGAAAAACAGATAATCGAATTGTGGCAGTGACAGTATATGCTGTTTTC

3*0 400 420 440 460 410

C G M T F P L L F E V Y K P R E B I , Q A G D K Y B T K P E I A A I L I K K L Q S

500 S20 540 560 5S0 600

M G F K F K L V L A D S L Y G E S G I C N F I S V L D E L N L N Y I V A I R S N H

^TGGGTTTTAAATTCAACTTAGTACTTGCAGATAGCTTATATGGAGAGAGTGGTAAGAATTTCATAtCTGTATTAGATGAACTAAACTTCAACTATATAGTAGCGATTCCGTCAAATCAT620 640 660 680 700 720

Y V E I L P R 0 H I Q Y I . K N Q K F 0 R V F S 0 L 5 R E N R F I R E I I P G K R

740 760 7*0 too 820 (40

G E L R Y M Q I T T D P E N L P D N T T B Y V M S K Y P D I T P R E V G N F Y GSGAGAACTTAGATATTGGCAAATTACTACAGATCCAGAAAATTTGCCTGATAACACTACTTGGTATGTGATGAGTAAATATCCAGACATTACGCCAAGAGAAGTTGGAAATTTTTACGGT

• 60 880 900 920 940 960

L R T W V E Y G L K Q S K N E L G I t S D F R L T H Y P D I E A M V G N Y L Q C L

980 1000 1020 1040 1060 tOlO

F N G V C I R S N C F S L H H H E S O N L f H I L G G I M e M A G R T F L T I F

l lCO i i a f i l l t O l l « O l l t O 1200

1220 1240 12€0

AGTTCAATTTTTATATCCCTGATTCACCCTGATTTCTACTTTTCCTCTGCCTAGAGTGACAAAAGAGGG1340 1360 TTR TJS9

\S702

AGAACTCTTGCAAAAGTCTTTTC

55 m 60

M S G V S F I Q T F H Y H V D V V K A D E K K K K K P G R R P K L 1 I E D Q V I . H

140 160 180 200 220 240

V I O Y H R E Y R T Y Y H I G L D H G L S E S A V C R T V Y K I E N I L I S S R

2S0 280 300 320 340 360

K F S L P G K K E L L K M P S Q E H L V V M D V T E S P I E R P K K S Q K H F F

380 400 420 440 460 480

5 G K A G E H T L K T Q L V I H 0 X T S 0 I I C L G H G K G R I H D F R L F K T

500 S20 540 560 5tO 600

S G V K F S E L L K V I A D K G Y Q G I T K I H K L S E T P I K K P K G K K L A

«Z0 6tO €60 ««0 100 120

K E Q K E Y N R E L N R L R I V V E H V N R R L K I F N I L S N Q Y R H R H R R

740 7C0 780 tOO 820 140

F G L R S H I i l A G I Y N Y E L A L K A *

860 Bao 900 920 940 960

CCATATCAAATTTTTGATTTAGCATTCTTGAGTATCAGTTAATATGAAAAATTTGGTATTCTCTGGCTATTTAAACTTACTCTAGCAGACAAATAGATAACCCTTTATTTTATCAAAAAA

980 1000 1020 1040 10 63 TBl?TATTTATGCAAGAGTTCT

Fig. 3. Nucleotide sequences of \S701 and \S702. and deduced amino acid sequences of their putative transposases. The underlined nucleotidesequences correspond to the left IR (IRJ and right IR (IRa) of both ISs. The amino acids of the \S701 transposase identical to those of transposasas fromtS23?, IS50RIS4, ISrOR, ISH7 and IS rS6 are underlined. The bracketed amino add sequence located upstream from the putative transposase of IS70?corresponds to the A/-terminal extension of the transposase if the translation is initiated at the GUG codon. =, sequences that are present in the IRL ofboth ISs and share homology with the - 3 5 and - 1 0 sequences defined tot RNA polymerase recognition in £. coJi; +. addrtionai identical nucleolides inthe same relative position in both ISs. These sequence data will appear in the EMBL/GenBank/DDBJ NucJeotide Sequence Data Ubrahes under theaccession numbers X60383 (\S701) and X60384 (IS703-

Cyanobacterial insertion sequences 2169

S 3I*, p-

5 3 z z z zkb

23 _

kb

23 ,

9.5.

e.9-

4.3-1

2.3.3 •

ti.

0.94^

Fig. 4. Autoradiograms of Southern blots of total DNAs from cyanobacterial strains after hybridization with a ̂ 'P-labelled DtMA probe internal to \B701 (A)and IS702 (B), All the cyanobacterial DNAs were digested by EcoRI and HincW. except those from Calothrix 7504 and 7601, wtiich were digested byWndlll. Tfie bars on the left show the positions of size markers generated by digestion of X DNA with H/ndlll. Genera are abbreviated as follows: C.Cylindrospermum: Ca. Calathtix: M. Microcystis: N, Nastoc\ Ps, Pseudanabaena: S, Synechococcus.

(Calothrix 7504 and 7601, Cylindrosperwum 73101, Nos-

toc 6720, 8009 and 73102). This may reflect the fact that

these strains share, or shared, the same habitats and that

they are. or were, able to exchange genetic material. This

hypothesis was first put forward to explain the occurrence

of plasmids sharing sequence homotogies in cyanobac-

teria belonging to different genera (Van den Hondel et ai,

1979; Lau eta/., 1980). However, the strains selected for

this study, and those that carry IS702-like sequences,

have been isolated from very distant countries: North

America, Indonesia, Australia or Sweden (Rippka et ai,

1979). Therefore the geographical link between these

different species, if any, must be very ancient or transfer

could have occurred more recently through intermediate

hosts.

Experimental procedures

Enzymes and chemicals

Restriction enzymes were purchased from either Boehn'nger orGenofit. [c.-32pj,^jcTP (3000 Ci mmol \ [a-^^SJ-dATP (1000 Cimmol '), Hybond N membrane and the nick translation kit v*/erefrom Amersham International. The Cyclone System used togenerate deletions was from International Biotechnology Inc. TheKiloBase Sequencing System was from Bethesda ResearchLaboratory. M13 derivatives, pTZ18R and pTZ19R, were pur-chased from Pharmacia. Enzymes were used according to themanufacturer's instructions. All chemicals were reagent-grade.

Cyanobacterial strains, culture conditions and DNA

extraction

All cyanobacterial strains were from the Pasteur Culture Collec-tion (PCC) and are referred to by their genus name followed bytheir number in the coHection, Culture conditions of Calothrix sp.strain PCC 7601 (previously called Fremyelta diplosiphon UTEX481) and the method for chromosomal DNA extraction have beendescribed previously (Mazel e/a/., 1986), Methods for chromoso-mal DNA extractions from the other cyanobaoteria were asdescribed by Damerval etai. (1989),

The mutants were isoiated during the course of transformationassays. Calothrix 7601 celts, after contact with pQSR49A (giftfrom J. Shapiro), were spread on selection plates containingeither ampicillin or streptomycin. Surviving colonies were devoidof exogenous plasmid, but several of them showed a colouredphenotype different from that of WT cells under the sameillumination (Tandeau de Marsac, 1983). Two main classes ofphenotyplcally related mutants were obtained, namely the GYclass (for Green-Yellow) and the BR class (for Brown-Red).

Library oonstruotions

Partial DNA libraries from Calothrix 7601 were constructed asfollows: total DNA was digested with EcoRI and fractionated byelectrophoresis on a 0.7% agarose gel. then DNA fragments ofappropriate sizes (see the Results) were eluted as describedpreviously (Vogelstein and Gillespie, 1979) and inserted into theEcoRI site of pTZlSR.

2170 D. Maze/etal.

DNA sequencing

DNA was cloned into either pTZ18R or pTZi9R for sequenceanalysis- Overlapping clones were obtained by using the CycloneSystem from IBI adapted to single-stranded pTZ18R as de-scribed previously (Mazei et ai, 1988)- Sequencing was carriedout by using the KiloBase Sequencing System from BRL onsingle-stranded DNA, using the Ml 3 reverse primer.

Hybridization with ^P-labelled probes

Nick translation and Southern hybridization experiments wereperformed as described previously fTandeau de Marsac et ai,1985), except that the nitrocellulose was replaced with Hybond Nmembranes. Hybridizations were performed at 65''C.

Acknowledgements

We thank Dr M- Herdman for careful reading of the manuscript.This work was supported by the Institut Pasteur and by the CentreNational de la Recherche Scientifique {URA1129),

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