The DevBCA exporter is essential for envelope
formation in heterocysts of the cyanobacterium
Anabaena
sp. strain PCC 7120
Gabriele Fiedler, Matthias Arnold, Stefan Hannus† and Iris Maldener*
Lehrstuhl fu¨r Zellbiologie und Pflanzenphysiologie, Universita¨t Regensburg, D-93040 Regensburg, Germany.
Summary
The gene devA of the filamentous heterocyst-form-ing cyanobacterium Anabaena sp. strain PCC 7120 encodes a protein with high similarity to ATP-binding cassettes of ABC transporters. Mutant M7 defective in thedevA gene is arrested in the development of het-erocysts at an early stage and is not able to fix N2 under aerobic conditions. ThedevA gene is differen-tially expressed in heterocysts. To gain a better under-standing of the structural components of this putative ABC transporter, we determined the complete nucleo-tide sequence of the entire gene cluster. The two addi-tional genes, nameddevB and devC, encode proteins with similarities to membrane fusion proteins (DevB) of several ABC exporters and to membrane-spanning proteins (DevC) of ABC transporters in general. Site-directed mutations in each of the three genes resulted in identical phenotypes. Heterocyst-specific glycolipids forming the laminated layer of the envelope were iden-tified in lipid extracts of M7 and in the site-directed mutants. However, transmission electron microscopy revealed unequivocally that the glycolipid layer is missing in mutant M7. Ultrastructural analysis also confirmed a developmental block at an early stage of differentiation. The results of this study suggest that thedevBCA operon encodes an exporter of glyco-lipids or of an enzyme that is necessary for the forma-tion of the laminated layer. The hypothesis is proposed that an intact envelope could be required for further heterocyst differentiation.
Introduction
The filamentous cyanobacteriumAnabaena spp. protects
the extremely oxygen-sensitive nitrogenase by spatially separating N2fixation and oxygenic photosynthesis in two
different cell types, the oxygen-evolving vegetative cell and the N2-fixing heterocyst. Upon deprivation of
inorgani-cally bound nitrogen, about 5–10% of the vegetative cells differentiate into heterocysts, resulting in a semi-regular spacing of these specialized cells along the trichome. Upon differentiation, morphological as well as biochemical changes in the developing heterocysts lead to the estab-lishment of a microaerobic environment tolerated by nitro-genase (reviewed in Haselkorn, 1978; Wolk, 1982; Fay, 1992; Gallon, 1992). A thick envelope is formed outside of the Gram-negative cell wall to reduce the diffusion of gases into the heterocyst (Walsby, 1985; Murry and Wolk, 1989). The inner laminated layer is composed of hetero-cyst-specific glycolipids, which are derivatives of hexoses containing long-chain polyhydroxylalcohols (Bryceet al., 1972). The outer homogeneous and probably the outer-most fibrous layer, too, are built of specific polysaccharides (Cardemil and Wolk, 1979). To minimize the diffusion of O2
into heterocysts from adjacent vegetative cells, the junc-tions between neighbouring cells are reduced to a narrow septum, which may be traversed by microplasmodesmata (Lang and Fay, 1971). Massive deposition of envelope material in this region leads to the formation of a pore channel. Close to the poles, the nitrogen storage mole-cule, cyanophycin, forms the so-called polar granule (Langet al., 1972; Ja¨ger et al., 1997). In addition to the cytoplasmic membranes and thylakoids, a third type of membrane is found close to the polar granule, forming the so-called honeycomb region (Lang and Fay, 1971; Braun-Howlandet al., 1988). In this area, light-indepen-dent oxidation of diaminobenzidine (DAB) can be obser-ved, indicating high concentrations of respiratory enzymes (Murry et al., 1981). Photosynthetic O2 production and
CO2fixation are restricted to vegetative cells. Therefore,
heterocysts depend on these cells for a supply of reduc-tant, probably delivered in the form of disaccharides (Schilling and Ehrnsperger, 1985). In return, heterocysts provide the vegetative cells with fixed nitrogen, presum-ably as glutamine (Thomaset al., 1977).
Only a few genes have been identified that are required for the formation of the heterocyst envelope. The hepA (formerhetA) gene functions in the stabilization or synth-esis of the polysaccharide layer (Holland and Wolk,
Q1998 Blackwell Science Ltd
Received 18 October, 1997; revised 19 December, 1997; accepted 23 December, 1997. †Present address: Universita¨t Heidelberg, Insti-tut fu¨r Biochemie I, Im Neunheimerfeld 328, D-69120 Heidelberg, Germany.*For correspondence. E-mail [email protected]; Tel. (941) 943 3033; Fax (941) 943 3352.
1990; Legane´s, 1994). Black et al. (1995) described mutant strain 543 of Anabaena 7120, in which the hglK gene had been inactivated. HglK might be involved in the formation of the glycolipid layer because, in the hglK mutant, heterocyst-specific glycolipids can be found in cell extracts, but the laminated layer is not formed. A gene cluster consisting ofhetM (or hglB ), hglC and hglD was identified that is required for heterocyst glycolipid synthesis as shown by mutational analysis (Black and Wolk, 1994; Baueret al., 1997; I. Maldener unpublished). Heterocyst differentiation is regulated at the level of transcription (Wolket al., 1994). The ntcA gene, encoding a global positive transcriptional regulator (Vega-Palaset al., 1992), can initiate a regulatory cascade of gene expres-sion upon nitrogen deprivation. A mutation ofntcA in Ana-baena 7120 blocks the differentiation of heterocysts and the expression of the early regulatory genehetR (Buikema and Haselkorn, 1991; Blacket al., 1993; Frı´as et al., 1994). A mutation inhetR blocks the expression of several later genes, e.g. thedevA gene described below (Black et al., 1993; Cai and Wolk, 1997).
Transposon mutagenesis ofAnabaena 7120 provides a useful tool for exploring the genes that are involved in heterocyst differentiation (Borthakur and Haselkorn, 1989; Wolk et al., 1991). Using this technique, many mutants which are unable to fix N2 under aerobic conditions,
owing to incompletely developed heterocysts, were iso-lated (Ernst et al., 1992). One of these mutants, M7, is arrested relatively early in the differentiation of hetero-cysts. The mutant is able to fix N2(Fixþ) under anaerobic
but not under aerobic (Fox¹) growth conditions. This defect is caused by an aberrant heterocyst envelope (Hen¹) and an arrest in protoplast maturation, which results in a lack of heterocyst-specific oxidation of DAB (Dab¹) (Ernstet al., 1992). Maldeneret al. (1994) showed that the pleiotropic phenotype of mutant M7 was caused by the transposition of Tn5-1063 into an open reading frame (ORF) named devA. Expression studies, using luxAB as a reporter, showed that devA expression increases approximately eightfold in whole filaments about 14 h after nitrogen stepdown; the increase in differentiating cells was even greater. The deduced amino acid sequence of DevA shows striking similarity to the ATP-binding subunit of ABC (ATP-binding cassette) transporters (Higgins et al., 1990). Transport systems of this family facilitate ATP-dependent transloca-tion of a great variety of substrates and are common in bacteria and in eukaryotes. Prokaryotic ABC transporters often comprise several subunits encoded by genes that are organized in an operon (Ames, 1986). The subunits include a periplasmic binding protein in the case of impor-ters or, in the case of several exporimpor-ters, a membrane fusion protein (MFP) working as a homodimer, connecting the outer and cytoplasmic membranes of Gram-negative bacteria. In addition to an ATP-binding subunit, in most
cases working as a homodimer, both types of transporters possess one or two membrane proteins that traverse the cytoplasmic membrane as a hetero- or homodimer (Ames, 1986; Dinhet al., 1994).
Speculating that thedevA gene is one component of an operon encoding the complete transporter, the DNA flank-ingdevA was analysed. A gene cluster of three genes, devB, devC and devA, was found possibly encoding an ABC exporter.Anabaena 7120 strains were constructed bearing an insertionally inactivateddevA, devB or devC gene respectively. The genotypical and phenotypical char-acterization of these mutants and the results of ultrastruc-tural analysis of mutant M7 by transmission electron microscopy are presented.
Results
The devBCA cluster
Figure 1 shows the map of thedevBCA gene cluster. Two genes which are located upstream ofdevA have the same orientation and form a gene cluster of 3540 bp including devA. The nucleotide sequences are available from the GenBank-EMBL database under accession number X99672. The first ORF upstream of devA is separated by 180 bp of a non-coding stretch. This ORF was named devC and comprises 1155 bp (it corresponds to orfA in Maldeneret al., 1994). The devC gene could encode a protein of 384 amino acids with a molecular mass of 43.4 kDa. Upstream of devC, spaced by a non-coding region of 41 bp, the first ORF of the gene cluster with 1425 bp,devB, was identified. The devB gene may encode a protein of 474 amino acids with a molecular mass of 51.6 kDa. The DNA sequence 1000 bp further upstream
Fig. 1. Restriction map of thedev region (4.4 kb) The three ORFs, devA , devB and devC, encoding the subunits of the presumptive ABC exporter are shown as filled arrows, indicating the direction of transcription. Restriction sites were mapped by sequencing, and all sites for each enzyme are shown. Sites of directed insertion of the cassette C.K3 and of transposition by Tn5-1063 are shown. The npt gene encoding neomycin resistance under the control of the psbA promoter was used as selection marker. The luxAB genes ser ved as reporters and are located at the left end of the transposon.
fromdevB does not reveal any ORF of significant size. A stem–loop structure, which may be part of a rho-indepen-dent signal for the termination of transcription, was irho-indepen-denti- identi-fied 33 bp downstream from thedevA stop codon.
Sequence comparisons
Besides a high similarity to the ATP-binding protein of importers, the sequence of DevA further shows extensive similarities to the ATP-binding subunits of several exporters (36% identity with the C-terminal half of HlyB, a haemolysin exporter ofEscherichia coli ).
The amino acid sequence comparison programBLASTP found membrane fusion proteins (MFPs) with similarity to the N-terminal part of the sequence of DevB (Fig. 2A).
Dinhet al. (1994) made a multiple alignment of various MFP sequences, which revealed a consensus sequence in the C-terminal part of these proteins (Fig. 2B). Table 1 summarizes the overall sequence similarity of DevB to prokaryotic MFPs.
The hydropathy profile of DevB fits the profile of typical MFPs (data not shown). DevB contains a hydrophilic region at the N-terminus (amino acids 1–20), followed by a highly hydrophobic part of about 20 residues that might be responsible for anchoring the MFP in the cytoplasmic membrane. Adjacent to the transmembrane stretch is a region of moderate hydrophobicity, followed by a strikingly hydrophilic part that possibly traverses the periplasmic space. The C-terminus consists of a slightly hydrophobic
b-strand, typical of outer membrane-associated domains
Fig. 2. Multiple alignment of (A) the conserved N-terminal regions and (B) the conserved C-terminal regions of DevB and several MFPs.
Abbreviations are described in Table 1. Asterisks indicate the identities of DevB to at least three of the aligned sequences derived from Dinh et al. (1994). The high percentage of conservative exchanges is not emphasized. Residue numbers for each protein are provided at the beginning and end of each line. The consensus sequence by Dinhet al. (1994) is shown below.
(Dinhet al., 1994). These data suggest that DevB is the membrane fusion protein of an ABC export system.
The hydropathy profile of the deduced amino acid sequence of DevC shows five significantly hydrophobic stretches of about 20 amino acids, which may form trans-membrane helices (data not shown); a similar profile with five helices was obtained with a different transmembrane prediction program (TMPP; Hofmann and Stoffel, 1992). DevC might be the integral membrane component of a putative ABC protein-mediated exporter.
Mutation of the dev gene cluster
Mutant M7 was reconstructed by directed mutagenesis of thedevA gene in the wild type of Anabaena 7120 using two different approaches. The first approach used the recovered transposon and flankingAnabaena DNA (plas-mid pRL1340) to create mutant DR238 (Maldeneret al., 1994). In the second approach, the devA gene cloned from a library of wild-type DNA as anSspI–DraI fragment in pIM13 (Maldeneret al., 1994) was mutagenized directly by the insertion of the kanamycin resistance cassette C.K3 (Elhai and Wolk, 1988a) into the uniqueNheI site (Fig. 1). After cloning into a suicide plasmid (pRL271; Blacket al., 1993), the interrupted gene (plasmid pIM22) was trans-ferred to the wild type ofAnabaena 7120 via conjugation; double recombinants (DR22) were obtained usingsacB as positive selection marker (Cai and Wolk, 1990). Five ran-domly chosen recombinants showed the same phenotype as mutant strains M7 and DR238 with the characteristics Fox¹, Hetþ, Dab¹and Hen¹, the last determined by light microscopy.
To check whether the threedev genes are functionally related,devB and devC were mutagenized directly by inser-tion of the C.K3 cassette in the sites shown in Fig. 1. The resulting constructs were transferred to wild-type Ana-baena 7120 on sacB containing suicide plasmids (details about the construction of plasmids is in Experimental
procedures). For each mutant strain, sucrose-resistant recombinants (DR42 mutated indevC or DR74 in devB respectively) were analysed and showed the same pheno-type as the devA mutants, i.e. Fox¹, Hetþ, Dab¹ and Hen¹. To confirm that the phenotype of devC mutant DR42 was caused by disruption ofdevC and not by repres-sion of thedevA gene by insertion of the cassette into the putative regulatory 58region ofdevA, an intact devA gene was transferred to thedevC mutant on a shuttle vector that could complement mutant M7 (pIM27 in Maldeneret al., 1994). This plasmid could not complement the devC mutant, showing that its phenotype was not caused by insufficient transcription of thedevA gene located down-stream.
An ORF, 400 bp downstream ofdevA, oriented in the opposite direction was identified (Maldeneret al., 1994) and named orf2 (accession no. X99672). The deduced amino acid sequence does not show similarity to other known sequences. An insertion mutant (DR29) was created using plasmid pIM29 (seeExperimental procedures). This mutant showed a phenotype that was indistinguishable from that of the wild type, being able to form mature hetero-cysts and to grow on N2as the sole source of nitrogen. The
genotype of each of the mutant strains was confirmed by Southern blot analysis (data not shown).
Analysis of heterocyst-specific glycolipids in the devBCAmutants
According to Ernstet al. (1992), mutant M7 lacks the het-erocyst-specific glycolipids (Hgl¹), as determined by thin-layer chromatography (TLC) of extracts of filaments that had been nitrogen star ved for 48 h. Cells of wild-type Ana-baena 7120, devC and devB mutants, as well as cells of mutant M7, were analysed in the same way. As shown in Fig. 3, in wild-type Anabaena 7120 and in each mutant that had been deprived of nitrogen, a spot was detected in a region that has been attributed to heterocyst-specific
Table 1. Comparison of the amino acid sequences of DevB and various membrane fusion proteins.
Protein Organism Similarity Identity
(MFP) (abbreviation) Substrate (%) (%) Reference
SapE Lactobacillus sake (Lsa) Sakacin A 43 22 Axelsson and Holck (1995)
ORF Haemophilus influenzae (Hin) Unknown 45 22 Fleischmannet al. (1995) PrtE Serratia marcescens (Sma) Metalloprotease 47 25 Le´toffe´et al. (1993)
LktD Actinobacillus actino- Leukotoxin 46 22 Guthmilleret al. (1990)
mycetemcomitans (Aac)
HlyD E. coli (Eco) Alpha-haemolysin 45 24 Schuleinet al. (1992)
SppE Lactobacillus sake (Lsa) Sakacin P 45 23 Holcket al. (unpublished) PrtE Erwinia chrysanthemi (Ech) Metalloprotease 51 24 Le´toffe´et al. (1990) AprE Pseudomonas aeruginosa (Pae) Alkaline protease 49 22 Duonget al. (1992) LktD Pasteurella haemolytica (Pha) Leukotoxin 45 22 Strathdee and Lo (1989) CyaD Bordetella pertussis (Bpe) Cyclolysin 44 25 Glaseret al. (1988) AppD Actinobacillus pleuropneumoniae (Apl) Haemolysin 43 22 Changet al. (1991)
CvaA E. coli (Eco) Colicin V 41 18 Gilsonet al. (1990)
glycolipids (Winkenbachet al., 1972), whereas this spot was not detected in extracts of cells grown on NO3¹(data
for the devB mutant are not shown). In extracts of the Hgl¹mutant, P2 (Ernstet al., 1992), the specific glycolipid spot did not appear after the induction of heterocyst forma-tion (Fig. 3). In conclusion, the phenotypes of M7, thedevB anddevC mutants have to be defined as Hglþ.
Ultrastructure of the heterocysts of mutant M7
Despite the presence of heterocyst-specific glycolipids in extracts of whole filaments, the heterocyst envelope of thedev mutants looks thin and less refractile under the light microscope (Ernstet al., 1992; Maldener et al., 1994). This prompted us to examine the ultrastructure of induced filaments of mutant M7 by electron microscopy. For the induction of heterocyst differentiation, NO3¹-grown
Ana-baena cultures were washed three times with NO3¹-free
medium and incubated for 48 h (M7) or 36 h (wild type) in the same medium. Figure 4 shows the ultrastructure of heterocysts of wild-type Anabaena 7120 and mutant
M7 after fixation with glutaraldehyde and permanganate. With this fixation procedure, the glycolipids are retained during dehydration (Lang and Fay, 1971; Winkenbachet al., 1972). Heterocysts of wild-type and mutant M7 possess the homogeneous layer consisting of polysaccharides. The innermost laminated layer composed of heterocyst-speci-fic glycolipids is completely absent in the mutant; in the wild type, however, this layer can be clearly seen near the junction between heterocyst and vegetative cell. The empty space between laminated layer and cell wall is an artefact that occurs frequently during preparation. The dis-tribution of intracytoplasmic membranes in heterocysts of mutant M7 is more confluent than in vegetative cells. How-ever, the formation of the honeycomb region near the poles does not take place. As expected for a Fix¹mutant, no polar granule is built up in the mutant, whereas the characteristic small septum at the junctions with vegeta-tive cells is clearly visible. Densely packed glycogen gran-ules can be seen in both cell types of the mutant, which may indicate a depletion of fixed nitrogen (Ernst et al., 1984); in the wild type, glycogen granules are present in
Fig. 3. Thin-layer chromatography of glycolipid extracts from cultures containing 50mg of chlorophyll. The position of heterocyst-specific glycolipids is indicated by an arrow. Glycolipids of NO3¹-deprived cells of wild type (1), mutant M7 (2) and DR42 (3). Glycolipids of NO3¹ -grown cells of wild type, in which heterocyst differentiation was not totally repressed (4), mutant M7 (5), DR42 (6), mutant P2 (7) and NO3
¹ -deprived cells of mutant P2 (8); mutant P2 was used as a Hgl¹
vegetative cells only. Vegetative cells of M7 cannot be distin-guished from vegetative cells of the wild type. In both strains, the large carboxysomes, containing the paracrystalline RUBISCO structure, are present in vegetative cells only.
Discussion
Three closely linked ORFs,devB, devC and devA, could form an operon encoding the subunits of an ABC pro-tein-mediated transporter. The relatively long non-coding sequences betweendevB and devC and between devC anddevA are not unusual for cyanobacterial operon struc-tures. Omataet al. (1993), for example, describe a gene cluster encoding an ABC transporter for NO3¹ with a
stretch of 197 bp between two of the genes that are tran-scribed polycistronically (see also Bartsevich and Pakrasi, 1995). To determine the total size of the transcript, North-ern blot analysis and reverse transcriptase–polymerase chain reaction (RT–PCR) were attempted intensively; but all efforts were without success, probably because of a very short half-time or low transcription rates (not shown). However, from the lack of any termination signals betweendevB, devC and devA, it could be predicted that the genes are transcribed polycistronically. The functional linkage between the threedev genes was shown by site-directed mutagenesis of each gene, resulting in mutants with identical phenotypes. These data are consistent with the idea that the three genes form an operon.
ThedevC-encoded protein shares the general size and hydropathy profile of typical integral membrane proteins of ABC transporters. The low similarity of the primary sequ-ence of DevC to other known membrane domains is not surprising, as sequence conservation of these components is very low. The ‘Dassa–Hofnung’ consensus sequence of membrane proteins of ABC importers is not present in ABC exporters and does not appear in the DevC sequence (Dassa and Hofnung, 1985). The sequence and hydropathy profile of DevB shows all the typical features of peptide or protein exporters described by Dinhet al. (1994). A signal peptide guiding the protein to the periplasmic space is present neither at the N-terminus of DevB nor in other MFPs. The presence of an MFP-like protein suggests that DevBCA belongs to the class of ABC protein-mediated exporters rather than to the class of ABC importers. A gene that could encode a periplasmic binding protein, an essential component of bacterial ABC importers, was not present in the neighbourhood of thedevBCA gene cluster. No gene for an additional component of the transporter or a putative gene that could encode the substrate of the ABC exporter is linked to thedevBCA exporter genes, as was found for some other MFP-coupled systems (Le´toffe´et al., 1996), e.g. for the haemoprotein exporter (hasDE ) of Ser-ratia marcescens (Le´toffe´ et al., 1994), for the haemolysin exporter (hlyBD ) of E. coli (Mackman et al., 1986) and for
Fig. 4. Transmission electron micrograph of ultrathin sections of
(A) a connection between a vegetative cell and a heterocyst of the wild type ofAnabaena 7120, (B) a heterocyst of mutant M7, and (C) a filament from which B was magnified. H, homogeneous layer; L, laminated layer; PN, polar granule. The bar represents 1mm. 1198 G. Fiedler, M. Arnold, S. Hannus and I. Maldener
the polysialic acid exporter (kpsMTE ) of E. coli (Bliss and Silver, 1996).
The MFPs of ABC exporters of carbohydrates, such as the polysialic acid exporter, do not show similarity to the MFPs of peptide/protein exporters (Bliss and Silver, 1996). Subunits of carbohydrate exporters are encoded by single genes, like the subunits of the DevBCA exporter. On the contrary, in the known peptide/protein exporters (with only one exception), membrane-spanning and ATP-binding domains reside on the same polypeptide (Fath and Kolter, 1993). Sequence similarity of the DevB protein to MFPs of peptide/protein exporters suggests a proteinaceous sub-stance as substrate of the DevBCA exporter. However, the structural organization of this exporter resembles that of the carbohydrate exporter. No predictions about the nature of the transported substrate of the DevBCA expor-ter can be made from the sequence comparison data.
Winkenbachet al. (1972) showed the identity of the het-erocyst-specific spot on TLCs (the hethet-erocyst-specific glyco-lipids) with the laminated layer of the envelope. The same lipids were found in extracts of thedevBCA mutants after nitrogen stepdown. However, envelope glycolipids cannot be assembled as a laminated layer outside the cell wall, as seen in electron microscopic sections. Our conclusion is that the DevBCA exporter translocates heterocyst-specific glycolipids or an enzyme, which might be required for the assembly of the laminated layer. The presence of the homogeneous layer in the heterocyst envelope of mutant M7 showed that the formation of the two layers is regu-lated independently.
The phenotype of thehglK mutant (Black et al., 1995) is similar to that of M7. However, in contrast to mutant M7, not only heterocysts but also vegetative cells of thehglK mutant were affected; structures similar to the ‘thylakoid lacunae’ described for thehglK mutant were not found in the heterocysts of mutant M7. Sequence analysis of HglK did not reveal similarities to known transport proteins. Therefore, we conclude thathglK and the devBCA cluster influence different aspects of the formation of the glyco-lipid layer.
The absence of the laminated layer serving as a primary barrier to the diffusion of oxygen (Murry and Wolk, 1989) explains well the Fox¹phenotype of thedevBCA mutants. The pleiotropic phenotype of mutant M7 could be explained by speculating that the maturation of the protoplast depends on the formation of the laminated layer. The profound changes in the distribution of intracytoplasmic membrane structures is blocked in mutant M7, resulting in a lack of the honeycomb region, which agrees well with the lack of DAB oxidation. The hypothesis is proposed that the establish-ment of a barrier to oxygen, resulting in a decreasedPO2,
could trigger the process of maturation. This hypothesis awaits proof in future experiments.
Experimental procedures Strains and growth conditions
Strains of Anabaena 7120 and derivatives (Table 3) were
grown under photoautotrophic conditions at 308C in the light as described previously (Ernstet al., 1992) with the following changes: dilution of liquid medium (A&A) was 1:4, and undi-luted medium was solidified with 1.5% agar. The mutant strains were grown in the presence of 5 mM NO3¹ and 50mg ml¹1
neomycin sulphate in liquid or 200mg ml¹1
in solidified medium. Medium for strain DR42 (pIM27) additionally contained strep-tomycin dihydrochloride and spectinomycin sulphate (2.5mg ml¹1
each). Heterocyst differentiation was induced by wash-ing the cultures three times with A&A/4 and resuspendwash-ing the filaments in the same medium. Chlorophyll content was estimated from methanolic extracts according to Mackinney (1941). Strains ofE. coli were grown on LB medium under standard conditions (Maniatiset al., 1982). Transfer of plasmids
by conjugation between Anabaena 7120 and E. coli was
achieved as described earlier using RP4 bearing strain J-53 and cargo strain HB101 bearing helper plasmid pRL528 in tri-parental matings (Wolket al., 1984; Elhai and Wolk, 1988b). Selection for recombinants was performed as described pre-viously (Cai and Wolk, 1990).
DNA isolation and analysis
Total DNA fromAnabaena strains was isolated as described previously (Cai and Wolk, 1990). Plasmids were purified
Table 2. Relevant plasmids used in this study.
Plasmid Marker Properties Reference
pIM11 BmrKmrSmr ClaI recovery of M7 This study pIM22 CmrEmrKmr C.K3 indevA in pRL271 This study
pIM27 SmrSpr devA on shuttle vector pRL1049 Maldeneret al. (1994) (Black and Wolk, 1994) pIM29 CmrEmrKmr C.K3 inorf2 in pRL271 This study
pIM42 CmrEmrKmr C.K3 indevC in pRL271 This study pIM74 CmrEmrKmr C.K3 indevB in pRL271 This study pRL271 CmrEmr Positive selection vector withsacB Blacket al. (1993) pRL448 AprKmr C.K3 source Elhai and Wolk (1988a) pRL1340 BmrKmrSmr EcoRV recovery of M7 Maldeneret al. (1994) pUC19 Apr pBR322-derived cloning vector Yanisch-Perronet al. (1985) Ap, ampicillin; Bm, bleomycin; Cm, chloramphenicol; Em, erythromycin; Km, kanamycin; Sm, streptomycin; Sp, spectinomycin.
from E. coli with the Qiagen-plasmid kits. Transposon Tn5-1063, together with flankingAnabaena DNA, was recovered from mutant M7 on aClaI fragment according to Wolk et al. (1991), creating plasmid pIM11. Sequencing was done with the T7 sequencing kit from Pharmacia using the M13 reverse and universal primers and oligonucleotides complementary to
theAnabaena DNA. Templates were derived from subclones
of pIM11 as described here:EcoRV fragments of pIM11 were cloned into theSmaI site of pUC19 resulting in pIM25 (1.7 kb insert) and pIM23 (3.6 kb insert). By digestion of pIM25 with XbaI or HincII and recircularization, pIM31 (1.5 kb fragment) and pIM33 (1.0 kb fragment) were created. pIM23 was sub-cloned by restriction withSapI, followed by mung bean nuclease treatment, digestion withHincII and cloning of the fragment into theSmaI site of pUC19. A 1.5 kb fragment of pIM23 was subcloned into theSmaI site of pUC19, and a 2.3 kb fragment was subcloned into theXbaI site of the same vector, giving pIM48 and pIM53. Sequence analysis was performed with theUWGCGpackage of the University of Wisconsin Genetics
Computer Group, version 7.3 (Devereuxet al., 1984). Sequence
comparisons were made using the BLASTP program of the
Heidelberg Unix Sequence Analysis Resources, version 4. Mutation of devA, devB, devC and orf2
Theorf2 gene on pRL1451 (Maldener et al., 1994) was dis-rupted by the insertion of C.K3, derived as aSmaI fragment
from pRL448 (Elhai and Wolk, 1988a), into the XmnI site
replacing a 45 bp fragment. After NdeI digestion, the insert was ligated into theAseI site of pRL271 (Black et al., 1993), resulting in pIM29. ThedevA gene was disrupted by the inser-tion of C.K3 into theNheI site, resulting in pIM16. An SphI– Ecl 136II fragment of pIM16 was ligated into the NruI and SphI sites of pRL271, creating pIM22. The devC gene derived from plasmid pIM11 was subcloned first as a 1.4- kbSpeI– HindIII fragment into pUC19 cut with XbaI and HindIII (pIM40). After the insertion of C.K3 into theEcoRV site of pIM40, the Ecl 136II–AseI fragment was ligated into pRL271, resulting in pIM42. To construct adevB mutant, plasmid pIM48 was diges-ted withSpeI, and C.K3, derived as an XbaI fragment from pRL448, was inserted. Insertion into pRL271 was achieved in the same way as with pIM42 creating pIM74.
Analysis of glycolipids
From 50 ml cultures that had been deprived of nitrogen for
48 h, total lipids were extracted twice with methanol–chloro-form (1:2). The extracts were evaporated in a stream of air, dissolved in 200ml of chloroform and chromatographed on thin-layer plates of silica gel (Merck) as described previously (Winkenbachet al., 1972).
DAB staining
DAB staining with 0.5 mg ml¹1diaminobenzidine was perfor-med after early log-phase cultures were deprived of nitrate for 24–48 h (Murryet al., 1981; Ernst et al., 1992).
Electron microscopy
Fixation with 2.5% glutaraldehyde and 2% KMnO4and
dehy-dration was performed as described previously (Blacket al., 1995). After dehydration, the samples were incubated in a 1:1 mixture of Durcupan (Fluka) and propylene oxide over-night at 378C, followed by embedding in Durcupan for 24 h at 378C and 48 h at 608C in BEEM capsules with an open lid. Thin sections of 70–90 nm were collected on copper grids and stained with uranyl acetate for 20 min and with lead citrate for 5 min. The samples were examined with a Zeiss EM109 electron microscope at 80 kV.
Acknowledgements
The authors would like to thank Professor Dr C. Peter Wolk, Michigan State University, for plasmids pRL271 and pRL448, Professor Dr Juergen Boeckh and Janna Streck from the Lehrstuhl fu¨r Zoologie, University of Regensburg for access to and assistance with electron microscopy and Dr Eckhard Loos and Dr Margret Sauter for critical reading of the manu-script. The work was supported by the Deutsche Forschungs-gemeinschaft (DFG).
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Strain Genotype Antibiotic resistance PCC 7120 Wild type M7 devA::Tn5-1063 Bm Nm Sm SR22 devA::C.K3 Cm Em Nm P2 hetM ::Tn5-1063 Bm Nm Sm DR22 devA::C.K3 Nm DR29 orf2 ::C.K3 Nm DR42 devC ::C.K3 Nm DR74 devB ::C.K3 Nm DR42 (pIM27) devC ::C.K3 Nm Sm Sp Nm, neomycin; DR, double recombinant; SR, single recombinant. Others as in Table 2.
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