Unité de Physiologie Microbienne (CNRS URA 1129), Département de Biochimie et Génétique Moléculaire, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France1
Author for correspondence: Isabelle Iteman. Tel: +33 1 45 68 84 16. Fax: +33 1 40 61 30 42. e-mail: iiteman{at}pasteur.fr
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ABSTRACT |
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Keywords: antitermination, cyanobacterium, ITS, rRNA, transfer RNA
Abbreviations: ITS, internal transcribed spacer; STRR, short tandemly repeated repetitive
The GenBank accession numbers for the sequences reported in this paper are AF180968 and AF180969 for ITS-L and ITS-S, respectively.
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INTRODUCTION |
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Cyanobacteria have colonized many ecological niches and have developed diverse strategies to survive in greatly different environments. Early endosymbiotic forms gave rise to the photosynthetic organelles (chloroplasts and cyanelles) of eukaryotes (see Giovannoni et al., 1988 ), thus sharing their ability to perform oxygenic photosynthesis. Extant free-living forms exhibit varied physiological properties and range in morphology from simple unicellular to complex filamentous organisms (Rippka et al., 1979
; Castenholz & Waterbury, 1989
). Under conditions of combined nitrogen limitation, many filamentous strains such as Nostoc PCC 7120 are able to differentiate heterocysts, specialized cells responsible for the aerobic fixation of molecular N2 (Wolk, 1996
). This morphological and physiological diversity is mirrored by extensive genetic variability, cyanobacterial genomes ranging in mean DNA base composition from 32 to 71 mol% G+C (Herdman et al., 1979b
; M. Herdman & R. Rippka, unpublished) and in complexity from 2·0 to 13·2 Mbp (Herdman et al., 1979a
; M. Herdman & R. Rippka, unpublished). Consequently, it cannot be excluded that transcriptional control and processing mechanisms may vary between different members of the cyanobacterial phylum. However, little is known about the organization of their rRNA operons. Limited data have been obtained from the physical or genetic maps of the unicellular strains Synechococcus PCC 6301 (Tomioka et al., 1981
), Synechococcus PCC 7002 (Chen & Widger, 1993
) and Synechocystis PCC 6803 (Kaneko et al., 1996
). Further information is available from the sequences of the 16S rRNA23S rRNA ITS domain in members of several cyanobacterial genera that include the unicellular Synechococcus PCC 6301 (Tomioka & Sugiura, 1984
), Synechocystis PCC 6803 (Kaneko et al., 1996
) and 47 strains of the genus Microcystis (Otsuka et al., 1999
), the filamentous (non-heterocystous) Arthrospira PCC 7345 (Nelissen et al., 1994
), Spirulina PCC 6313 (Nelissen et al., 1994
) and Trichodesmium NIBB 1067 (Wilmotte et al., 1994
), the heterocystous Nodularia BCNOD 9427 (Hayes & Barker, 1997
) and a strain of uncertain generic identity, Mastigocladus HTF strain PCC 7518 (G. Van der Auwera & A. Wilmotte, personal communication). In addition, the sequence of the ITS of the photosynthetic cyanelle of Cyanophora paradoxa has been determined (Janssen et al., 1987
).
The cyanobacterial ITS regions investigated to date vary in size from 354 to 545 nucleotides (287 in the cyanelle) and, for those that have been sequenced, only a single ITS species was found in each strain. This is surprising, since sequence and length information on the 16S rRNA23S rRNA spacer in other bacterial groups suggests that considerable variation can occur not only between species but also between the alleles of the rRNA operon within a single strain (Gürtler & Stanisich, 1996 ). Consistent with other investigations (Lu et al., 1997
; Neilan et al., 1997
), we have indeed observed multiple ITS products in PCR amplifications of many cyanobacteria, in particular among the filamentous heterocystous strains (Iteman et al., 1999
, and unpublished data). However, no comparative analyses are available for the ITS regions that have been sequenced so far, and the differences between spacer regions of different lengths within a single cyanobacterial genome have not been examined. Furthermore, the regions involved in coordinated transcription of cyanobacterial rrn operons and maturation of their products have not yet been identified, and anti-terminator box Bbox A motifs have been reported to be undetectable in Synechococcus PCC 6301 (Berg et al., 1989
). We have therefore sequenced and analysed the PCR products corresponding to two 16S rRNA23S rRNA spacer domains of different sizes in Nostoc PCC 7120, which contains four rrn operons (Ligon et al., 1991
). The two distinct sequences have been aligned with a selection of those available from other cyanobacteria, E. coli and the photosynthetic cyanelle of C. paradoxa. The putative secondary structures of some deduced rRNA transcripts have been compared, and anti-terminator and structural motifs potentially implicated in the control of rrn transcription and maturation in cyanobacteria have been identified.
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METHODS |
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Amplification of the ITS regions.
PCR amplifications were performed with DNA of Nostoc PCC 7120 and Synechocystis PCC 6803 isolated by the mini extraction method (Cai & Wolk, 1990 ) or directly with cell lysates obtained by five alternating cycles of freezing in liquid nitrogen and thawing at 50 °C. A set of primers (322 and 340) designed initially for sequencing was used to specifically amplify the part of the rRNA operon containing the ITS region. The sequence and the position of each primer are indicated in Fig. 1
. The PCR mixture contained 10 µl Taq (10x) commercial buffer, 10 µl lysate or purified DNA (100500 ng), 150 µM of each dNTP, 500 ng of each primer and 2·5 U Taq polymerase (Appligene). The total reaction volume was 100 µl. After an initial cycle consisting of 3 min at 95 °C, 2 min at 55 °C and 30 s at 72 °C, 30 cycles of amplification were started (1·5 min at 95 °C, 2·5 min at 55 °C and 3 min at 72 °C). The termination cycle was 7 min at 72 °C. The PCR products were migrated either on 1·5% (w/v) agarose gels or on denaturing gels containing 8% (w/v) polyacrylamide and urea (6 M) in 0·25xTris/borate/EDTA buffer (Sambrook et al., 1989
), and visualized by staining with ethidium bromide.
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Sequence alignment.
The ITS sequences of Nostoc PCC 7120 were first aligned to a selection of the available ITS sequences of free-living cyanobacteria, the cyanelle of C. paradoxa and the rrnA operon of E. coli by large block identity using the software Macaw v. 2.05 (Multiple Alignment Construction and Analysis Workbench, NCBI). The alignment was then manually refined using Genedoc v. 2.4 (Nicholas & Nicholas, 1997 ) by reference to our secondary structure models (see below). The aligned sequences (Fig. 3
), with GenBank accession numbers in parentheses, are: PCC 7120, Nostoc PCC 7120 (this study); BCNOD 9427, Nodularia BCNOD9427 (AJ224448); PCC 7518, Mastigocladus HTF strain PCC 7518 (G. Van der Auwera & A. Wilmotte, personal communication); PCC 6313; Spirulina PCC 6313 (X75045); NIBB1067, Trichodesmium NIBB1067 (X72871); PCC 7345, Arthrospira PCC 7345 (X75044); PCC 6301, Synechococcus PCC 6301 (K01983); PCC 6803, Synechocystis PCC 6803 (D90916); TC8, Microcystis TC8 (AB015386); Cyanelle, Cyanophora paradoxa cyanelle (M19493); E. coli, E. coli K-12 MG1655 rrnA operon (AE000460, AE000461). The sequence of Microcystis strain TC8 was chosen as representative of the 47 virtually identical (Otsuka et al., 1999
) sequences currently available.
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RESULTS |
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Secondary structure of putative rrn transcripts
With the exception of Nostoc PCC 7120, for which only the sequence of the 16S rRNA23S rRNA ITS region is available, the secondary structures of the transcripts (Fig. 4a, b
) commence at the putative transcription start site of the promoter P1 and include the 5' leader sequence upstream from the 16S rRNA, plus the 16S rRNA23S rRNA ITS and the 23S rRNA5S rRNA spacer. Despite large variations in the length of the transcripts, the secondary structures show many highly conserved regions (see also Fig. 3
). Near the 5' end of the transcripts, a stem that carries the 16S rRNA is formed by pairing of part of the ITS with the 5' leader sequence of the operon (Fig. 4a
, b
). In all organisms examined, this stem has a conserved domain (D2) at the base. However, the remainder of this promoter region of the operon is highly variable (Fig. 4a
, b
), since it contains one to three stemloop structures (region V1). At the beginning of the ITS, immediately following the 16S rRNA, a conserved domain (D1) containing 9 bases is observed in all free-living cyanobacterial strains examined (Fig. 3
). This domain pairs both with part of the V1 region preceding the 16S rRNA, and with a second conserved domain (D1') of the ITS, to form the basal part of a stemloop structure (Fig. 4a
, b
). This stem, except in the D1/D1' region, is highly variable and ranges in length from a minimum of 35 bases in the cyanelle to 85 bases in Spirulina PCC 6313 (Fig. 3
). In E. coli, the sequence corresponding to D1 is entirely paired with a 5' leader region (Fig. 4a
).
The unpaired domain situated between D2 and the tRNA region contains a short conserved sequence (D3) flanked upstream and downstream by variable-sized regions of 235 bases and 426 bases, respectively (Fig. 3). The stem V2 (Figs 3
, 4a
and 4b
) observed immediately after the tRNAIle in Nostoc PCC 7120 (ITS-L), Spirulina PCC 6313 and Synechococcus PCC 6301 varies in length from 24 to 96 bases and, although shorter, is also present in the E. coli sequence. In Nostoc PCC 7120, this stem is composed of four copies of short tandemly repeated repetitive (STRR) sequences: 5'-G(A/T)(C/T)AAAA-3' in the 5' part of the helix and complementary 5'-TTTTG(A/G)A-3' sequences in the 3' part. The tRNA domain is followed by conserved antiterminator regions (boxes A and B; see below) and then by another stem that carries the 23S rRNA and associated structures. This processing stem is formed by pairing of the ITS with part of the 23S rRNA5S rRNA spacer region and has a highly conserved domain (D4) at the base (Fig. 4a
, b
). Domain D5, situated at the 3' extremity of the ITS and immediately preceding the 23S rRNA, is less conserved than the D1/D1' region adjacent to the 16S rRNA and, as in E. coli, is paired with part of the 23S rRNA5S rRNA spacer (Fig. 4a
). The D5 region is preceded in 8 of the 12 sequences examined by a variable stemloop structure (V3) formed entirely by pairing between parts of the ITS and containing up to 108 bases; this is absent from Mastigocladus HTF strain PCC 7518, Arthrospira PCC 7345, Microcystis TC8 and the cyanelle (Fig. 3
).
Antitermination sequences and maturation sites
The bacterial rrn operon normally contains antiterminator sites that are essential for maintaining transcription of the rRNA genes in the correct stoichiometry (Condon et al., 1995 ); box A sequences occur either within each of the two processing stems or immediately adjacent to them, and in both cases they are preceded by a box B stem structure. By comparison of the secondary structures (Fig. 4a
, b
), we identified a motif of 12 nucleotides in the cyanobacterial spacer regions that corresponds to antiterminator box A. This is immediately adjacent to domain D4 at the base of the 23S rRNA processing stem in all sequences analysed and is extremely well conserved in the free-living cyanobacteria (consensus 5'-GAACCTTGAAAA-3') but differs in the cyanelle of C. paradoxa (Fig. 3
). Box A is immediately preceded in all cases, except the cyanelle, by a stemloop structure equivalent to box B of E. coli, situated 764 bases 3' to the tRNA (Figs 3
, 4a
and 4b
). We also found a motif corresponding to box A in the leader region 5' to the 16S rRNA of Synechococcus PCC 6301, Synechocystis PCC 6803 and the cyanelle, the only organisms for which the sequence of this region is available. As for the spacer box A, that of the leader region is similar in sequence (5'-GA/GACCUAGACAA-3') in the free-living cyanobacteria but differs in the cyanelle (Fig. 4a
, b
). The box B of the leader was deduced uniquely from its structure and position relative to box A since, as in other bacteria (Berg et al., 1989
), there was no homology in sequence between this domain in the spacer and leader regions.
Following transcription, the pre-mature rRNAs and mature tRNAs are released by cleavage involving enzymes RNase III and RNase P, respectively (King et al., 1986 ). The cleavage sites of RNase III are known in E. coli (Fig. 4a
) and we have positioned the hypothetical sites in the cyanobacterial transcripts where possible. This was feasible for Synechococcus PCC 6301 for both 16S rRNA and 23S rRNA (Fig. 4a
); for the cyanelle an apparent site was found only for the 23S rRNA (Fig. 4b
). No potential RNase III sites could be identified in the other cyanobacterial sequences. The presumptive cleavage sites of RNase P are indicated for all sequences in Fig. 4(a
, b
).
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DISCUSSION |
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ITS-L and ITS-S are virtually identical in sequence along their entire length (Fig. 3) and in secondary structure (not shown), except in the regions encoding tRNAIle and tRNAAla which are absent from the shorter ITS-S (Fig. 3
). Multiple rrn operons containing ITSs of different sizes have also been observed in other bacteria, and the ITSs of the different operons within a single organism commonly differ in their content of tRNA genes (Gürtler & Stanisich, 1996
). However, in contrast to Nostoc PCC 7120, genome sequencing has revealed only two copies of the rrn operon in the C. paradoxa cyanelle (GenBank accession number U30821) and in Synechocystis PCC 6803 (D64000 and D90916). The ITSs within each copy are identical in length and in sequence, and the two copies therefore encode the same tRNA gene(s). Similarly, the two ITS regions of Synechococcus PCC 6301 show only six differences in the 395 nucleotides (representing 72% of the sequence and including both tRNA genes) available for comparison (Williamson & Doolittle, 1983
; Tomioka & Sugiura, 1984
).
It is not clear whether the two tRNA genes of Nostoc PCC 7120 have been acquired by ITS-L in the course of evolution or lost from ITS-S. In either case, however, two independent insertion or deletion events must have occurred, since both types of ITS share a part of the V2 structure (positions 401440 of the alignment, Fig. 3). The formation of ITS-S from ITS-L by developmentally regulated excision events, such as occur in the nif operon during heterocyst differentiation in response to nitrogen starvation (Golden et al., 1985
, 1988
), can be excluded on two grounds. Firstly, both ITS-L and ITS-S were present in the same stoichiometry in cells of Nostoc PCC 7120 cultivated in both the presence and absence of combined nitrogen; secondly, the 401440 region of ITS-S shows only 59% identity to that of ITS-L, suggesting a relatively ancient divergence, whereas excision events on either side of this region would not result in extensive sequence variation. The different ITS regions, although widespread in heterocystous cyanobacteria (Iteman et al., 1999
), are thus unlikely to have been formed as a consequence of cellular differentiation. Provided that the transcripts are sufficiently stable, it will be of interest to determine whether expression of the two types of ITS is differentially regulated in response to nutritional or developmental stimuli.
Comparison of the cyanobacterial ITS sequences shows that they are very variable in size (283545 nucleotides). However, their length does not correlate with the presence or absence of the tRNA genes. For example, the ITS of Nodularia BCNOD 9427 lacks both tRNA genes but is similar in size (354 nucleotides) to that of Mastigocladus HTF strain PCC 7518 (382 nucleotides) that contains both genes and is larger than the ITS-S of Nostoc PCC 7120 (283 nucleotides) that lacks both genes. These discrepancies are the result of large differences in the length of the variable regions, particularly of region V3. The cyanelle of C. paradoxa is considered as an intermediary stage in the evolution of chloroplasts from an endosymbiotic cyanobacterium (Herdman & Stanier, 1977 ; Aitken & Stanier, 1979
). Although the ITS of the cyanelle contains only 287 nucleotides, both tRNA genes are present; however, the stemloop structure following the 16S rRNA gene is short and regions V2, V3 and box B of the spacer are absent (Fig. 4b
). A similar organization is found in the chloroplast of the red alga Porphyra (data not shown), whose ITS has a length of 279 nucleotides. The cyanelle and chloroplast therefore probably represent the shortest cyanobacterial-type ITS that contains both tRNA genes. The absence of tRNA genes from the ITS-S of Nostoc PCC 7120 and from the ITSs of several other cyanobacteria is clearly not deleterious to the organisms, since a similar situation is often found in other bacteria (Gürtler & Stanisich, 1996
). In addition, genome sequencing (Kaneko et al., 1996
) has shown that the tRNAIle gene of Synechocystis PCC 6803 is present not only in both rrn operons but also as a third copy elsewhere in the genome, and that the absence of the tRNAAla gene from both operons is compensated by the presence of three copies at other locations.
Irrespective of the length of the ITS region, numerous short domains are conserved in all cyanobacteria examined, and most of them are homologous to those observed in E. coli. Regions D1, D2, D4 and D5 are required for the correct folding of the rRNA transcript (Fig. 4a, b
), the D2 and D4 motifs being of particular importance in contributing to the formation of the double-stranded 16S rRNA and 23S rRNA processing stems, respectively. Correct secondary structure is indispensable for the pre-maturation of the 16S rRNA and 23S rRNA molecules by RNase III (Srivastava & Schlessinger, 1990
). Although we identified putative RNase III cleavage sites in the 16S rRNA processing stem of Synechococcus PCC 6301 and in region D5 of the latter and of the cyanelle of C. paradoxa, we were unable to locate such sites in the sequences of Nostoc PCC 7120 and Synechocystis PCC 6803. Since a gene encoding RNase III exists in the latter organism (Kaneko et al., 1996
), our sequence data indicate that, as in other bacteria (see Nicholson, 1999
), this enzyme is most probably a sequence-nonspecific nuclease. In other bacteria, the antiterminator box Bbox A regions are involved in maintaining the correct stoichiometry of transcription of the rRNA genes (Berg et al., 1989
; Condon et al., 1995
). Antiterminator sequences were previously thought to be absent from Synechococcus PCC 6301 (Berg et al., 1989
), although Wilmotte et al. (1994)
suggested that a conserved region identified in the ITS of Synechococcus PCC 6301, Trichodesmium NIBB 1067 and seven other cyanobacteria (genera and strain numbers were not provided) may be involved in processing of the rRNA precursor. We have shown in this communication that the latter motif corresponds to the antiterminator box A and, together with box B, is present in the unpaired leader and spacer regions of all cyanobacterial sequences examined. As in E. coli, the two box A sequences are located adjacent to, and not within, the 16S rRNA and 23S rRNA processing stems and are preceded by a box B stem structure (Fig. 4a
, b
). However, box B is absent from the spacer of the cyanelle of C. paradoxa, and may thus not be essential for the control of transcription. This is consistent with studies involving site-directed mutagenesis of the antiterminator region in E. coli, which suggested that only box A has an essential role (Gourse et al., 1986
). The only major difference in secondary structure of the cyanobacterial and E. coli transcripts involves region D1, which is entirely paired with the leader sequence of the transcript in E. coli, but pairs with region D1' of the ITS to form a stemloop structure in the cyanobacteria examined (Fig. 4a
, b
). The similarity of essential regulatory and structural motifs in organisms as divergent as cyanobacteria and Proteobacteria suggests that mechanisms involved in the coordinated transcription and processing of the rRNA genes have been conserved throughout the course of evolution.
An unusual feature in the ITS-L of Nostoc PCC 7120 is that the V2 structure is composed of complementary paired STRR elements. STRR sequences appear to be common in heterocystous cyanobacteria, where they occur primarily, though not exclusively, in intergenic regions (Jackman & Mulligan, 1995 ; Mazel et al., 1990
; Vioque, 1997
), but they are rarely paired with a complementary sequence and have not been reported previously in the rrn operon. At least nine different STRR families are known (Jackman & Mulligan, 1995
). The Nostoc PCC 7120 STRR sequences within the ITS are similar to STRR type 6 sequences that form a similar structure (Vioque, 1997
) within the Rnase P transcript of another Nostoc strain, PCC 7937 (Anabaena ATCC 29413), but differ from those found (Mazel et al., 1990
) elsewhere in the Nostoc PCC 7120 genome. Since STRR sequences are absent from the ITSs of all other cyanobacteria examined, and stem V2 is missing from many, it would appear that this region serves no essential function. Site-directed mutagenesis of the similar region in the gene encoding RNase P led to the same conclusion (Vioque, 1997
).
tRNAIle and tRNAAla are the only tRNA genes identified to date in cyanobacterial rrn operons. The percentage of identity of each of these genes in the strains analysed is very high (Fig. 3). With the exception of the tRNAAla gene of Synechococcus PCC 6301, the cyanobacteria and the cyanelle do not encode the 3'-terminal CCA extension found in many bacteria (including E. coli; Fig. 3
), Archaea and eukaryotes (see Dirheimer et al., 1995
). However, they contain a characteristic subterminal CCA sequence at the 3' end of the stem that may be easily mistaken for this extension. When not encoded, the 3'-terminal CCA extension, which is also absent from chloroplast tRNA genes (Martin, 1995
), is most likely added post-transcriptionally, as in the latter, by ATP(CTP):tRNA nucleotidyltransferase (Martin, 1995
) to form the mature tRNA.
The size and number of ITS bands recovered by PCR amplification will be valuable for the genetic classification of cyanobacterial strains when a sufficiently large database has been established. However, we have shown unequivocally (Fig. 2) that, if a strain contains several different ITS regions, bands corresponding to heteroduplexes may be produced. Such heteroduplexes have also been described in other bacteria, their formation being influenced by the PCR protocols employed (Jensen et al., 1993
). The interpretation of multiple products should therefore be made with caution, and they are best verified by denaturing gel electrophoresis. The successful alignment of cyanobacterial ITS regions (Fig. 3
) may be exploited for the rapid incorporation of new sequences, the choice of restriction sites for RFLP analysis of unknown strains, and the design of probes and primers suitable for in situ hybridization and for PCR amplification. However, target regions resembling STRR sequences should be avoided, since these have been found at different locations in the genome (Mazel et al., 1990
; Vioque, 1997
) and do not appear to be specific to a given organism. Cyanobacterial-specific probes can be developed from the conserved motifs of the ITS, whereas several of the variable regions appear to be useful for discrimination at higher taxonomic resolution.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bensaadi-Merchermek, N., Salvado, J.-C., Cagnon, C., Karama, S. & Mouchès, C. (1995). Characterization of the unlinked 16S rDNA and 23S5S rRNA operon of Wolbachia pipientis, a prokaryotic parasite of insect gonads.Gene 165, 81-86.[Medline]
Berg, K. L., Squires, C. & Squires, C. L. (1989). Ribosomal RNA operon anti-termination. Function of leader and spacer region box Bbox A sequences and their conservation in diverse micro-organisms.J Mol Biol 209, 345-358.[Medline]
Birnboim, H. C. & Doly, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA.Nucleic Acids Res 7, 1513-1523.[Abstract]
Cai, Y. & Wolk, C. P. (1990). Use of a conditionally lethal gene in Anabaena sp. strain PCC 7120 to select for double recombinants and to entrap insertion sequences.J Bacteriol 172, 3138-3145.[Medline]
Castenholz, R. W. & Waterbury, J. B. (1989). Group 1. Cyanobacteria. In Bergeys Manual of Systematic Bacteriology, pp. 1710-1727. Edited by J. T. Staley, M. P. Bryant, N. Pfennig & J. G. Holt. Baltimore: Williams & Wilkins.
Chen, X. & Widger, W. R. (1993). Physical genome map of the unicellular cyanobacterium Synechococcus sp. strain PCC 7002.J Bacteriol 175, 5106-5116.[Abstract]
Condon, C., Squires, C. & Squires, C. L. (1995). Control of rRNA transcription in Escherichia coli.Microbiol Rev 59, 623-645.[Abstract]
De Rijk, P. & De Wachter, R. (1997). RnaViz, a program for the vizualisation of RNA secondary structure.Nucleic Acids Res 25, 4679-4684.
Dirheimer, G., Keith, G., Dumas, P. & Westhof, E. (1995). Primary, secondary and tertiary structures of tRNAs. In tRNA: Structure, Biosynthesis, and Function, pp. 93-126. Edited by D. Söll & U. RajBhandary. Washington, DC: American Society for Microbiology.
Fukunaga, M. & Mifuchi, I. (1989). Unique organization of Leptospira interrogans rRNA genes.J Bacteriol 171, 5763-5767.[Medline]
Giovannoni, S. J., Turner, S., Olsen, G. J., Barns, S., Lane, D. J. & Pace, N. R. (1988). Evolutionary relationships among cyanobacteria and green chloroplasts.J Bacteriol 170, 3584-3592.[Medline]
Golden, J. W., Robinson, S. J. & Haselkorn, R. (1985). Rearrangement of nitrogen fixation genes during heterocyst differentiation in the cyanobacterium Anabaena.Nature 314, 419-423.[Medline]
Golden, J. W., Carrasco, C. D., Mulligan, M. E., Schneider, G. J. & Haselkorn, R. (1988). Deletion of a 55-kilobase-pair DNA element from the chromosome during heterocyst differentiation of Anabaena sp. strain PCC 7120.J Bacteriol 170, 5034-5041.[Medline]
Gourse, R. L., deBoer, H. A. & Nomura, M. (1986). DNA determinants of rRNA synthesis in E. coli: growth rate dependent regulation, feedback inhibition, upstream activation, antitermination.Cell 44, 197-205.[Medline]
Gürtler, V. & Stanisich, V. A. (1996). New approaches to typing and identification of bacteria using the 16S23S rDNA spacer region.Microbiology 142, 3-16.[Medline]
Hayes, P. K. & Barker, G. L. A. (1997). Genetic diversity within Baltic Sea populations of Nodularia (Cyanobacteria).J Phycol 33, 919-923.
Herdman, M. & Stanier, R. Y. (1977). The cyanelle: chloroplast or endosymbiotic prokaryote?FEMS Microbiol Lett 1, 7-12.
Herdman, M., Janvier, M., Rippka, R. & Stanier, R. Y. (1979a). Genome size of cyanobacteria.J Gen Microbiol 111, 73-85.
Herdman, M., Janvier, M., Waterbury, J. B., Rippka, R., Stanier, R. Y. & Mandel, M. (1979b). Deoxyribonucleic acid base composition of cyanobacteria.J Gen Microbiol 111, 63-71.
Iteman, I., Rippka, R., Tandeau de Marsac, N. & Herdman, M. (1999). Use of molecular tools for the study of genetic relationships of heterocystous cyanobacteria. In Marine Cyanobacteria (Bulletin de lInstitut Océanographique, Monaco, special issue 19), pp. 13-20. Edited by L. Charpy & A. Larkum. Monaco: Institut Océanographique.
Jackman, D. M. & Mulligan, M. E. (1995). Characterization of a nitrogen-fixation (nif) gene cluster from Anabaena azollae 1a shows that closely related cyanobacteria have highly variable but structured intergenic regions.Microbiology 141, 2235-2244.[Abstract]
Janssen, I., Mucke, H., Löffelhardt, W. & Bohnert, H. J. (1987). The central part of the cyanelle rDNA unit of Cyanophora paradoxa: sequence comparison with chloroplasts and cyanobacteria.Plant Mol Biol 9, 479-484.
Jensen, M. A., Webster, J. A. & Straus, N. (1993). Effect of PCR conditions on the formation of the heteroduplex and single-stranded DNA products in the amplification of bacterial ribosomal DNA spacers regions.PCR Methods Appl 3, 186-194.[Medline]
Kaneko, T., Sato, S., Kotani, H. & 21 other authors (1996). Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA Res 3, 109136.[Medline]
King, T. C., Sirdeskmukh, R. & Schlessinger, D. (1986). Nucleolytic processing of ribonucleic acid transcripts in procaryotes.Microbiol Rev 50, 428-451.
Lachance, M. A. (1981). Genetic relatedness of heterocystous cyanobacteria by deoxyribonucleic aciddeoxyribonucleic acid reassociation.Int J Syst Bacteriol 31, 139-147.
Ligon, P. J. B., Meyer, K. G., Martin, J. A. & Curtis, S. E. (1991). Nucleotide sequence of a 16S rRNA gene from Anabaena sp. strain PCC 7120.Nucleic Acids Res 19, 4553.[Medline]
Lu, W., Evans, H. E., McColl, M. & Saunders, V. A. (1997). Identification of cyanobacteria by polymorphisms of PCR-amplified ribosomal DNA spacer region.FEMS Microbiol Lett 153, 141-149.
Martin, N. (1995). Organellar tRNAs: biosynthesis and function. In tRNA: Structure, Biosynthesis, and Function, pp. 127-140. Edited by D. Söll & U. RajBhandary. Washington, DC: American Society for Microbiology.
Matzura, O. & Wennborg, A. (1996). RNAdraw: an integrated program for RNA secondary structure calculation and analysis under 32-bits Microsoft Windows.CABIOS 12, 247-249.[Abstract]
Mazel, D., Houmard, J., Castets, A.-M. & Tandeau de Marsac, N. (1990). Highly repetitive DNA sequences in cyanobacterial genomes.J Bacteriol 172, 2755-2761.[Medline]
Naïmi, A., Beck, G. & Branlant, C. (1997). Primary and secondary structures of rRNA spacer regions in enterococci.Microbiology 143, 823-834.[Abstract]
Neilan, B. A., Stuart, J. L., Goodman, A. E., Cox, P. T. & Hawkins, P. R. (1997). Specific amplification and restriction polymorphisms of the cyanobacterial rRNA operon spacer region.Syst Appl Microbiol 20, 612-621.
Nelissen, B., Wilmotte, A., Neefs, J.-M. & De Wachter, R. (1994). Phylogenetic relationships among filamentous helical cyanobacteria investigated on the basis of 16S ribosomal RNA gene sequence analysis.Syst Appl Microbiol 17, 206-210.
Nicholas, K. B. & Nicholas, H. B., Jr (1997). Genedoc: a tool for editing and annotating multiple sequence alignments. www.cris.com/~ketchup/genedoc.shtml.
Nicholson, A. W. (1999). Function, mechanism and regulation of bacterial ribonucleases.FEMS Microbiol Rev 23, 371-390.[Medline]
Ojaimi, C., Davidson, B. E., Saint Girons, I. & Old, I. G. (1994). Conservation of gene arrangement and an unusual organization of rRNA genes in the linear chromosomes of the Lyme disease spirochaetes Borrelia burgdorferi, B. garinii and B. afzelii.Microbiology 140, 2931-2940.[Abstract]
Otsuka, S., Suda, S., Li, R. H., Watanabe, M., Oyaizu, H., Matsumoto, S. & Watanabe, M. M. (1999). Phylogenetic relationships between toxic and non-toxic strains of the genus Microcystis based on 16S to 23S internal transcribed spacer sequence.FEMS Microbiol Lett 172, 15-21.[Medline]
Rippka, R. & Herdman, M. (1992). Catalogue of Strains. Pasteur Culture Collection of Cyanobacterial Strains in Axenic Culture. Paris: Institut Pasteur.
Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M. & Stanier, R. Y. (1979). Generic assignments, strain histories and properties of pure cultures of cyanobacteria.J Gen Microbiol 111, 1-61.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Srivastava, A. K. & Schlessinger, D. (1990). Mechanism and regulation of bacterial ribosomal RNA processing.Annu Rev Microbiol 44, 105-129.[Medline]
Tomioka, N. & Sugiura, M. (1984). Nucleotide sequence of the 16S23S spacer region in the rrnA operon from a blue-green alga, Anacystis nidulans.Mol Gen Genet 193, 427-430.
Tomioka, N., Shinozaki, K. & Sugiura, M. (1981). Molecular cloning and characterization of ribosomal RNA genes from a blue-green alga, Anacystis nidulans.Mol Gen Genet 184, 359-363.
Vioque, A. (1997). The RNase P RNA from cyanobacteria: short tandemly repeated repetitive (STRR) sequences are present within the RNase P RNA gene in heterocyst-forming cyanobacteria.Nucleic Acids Res 25, 3471-3477.
Williamson, S. E. & Doolittle, W. F. (1983). Gene for tRNAIle and tRNAAla in the spacer between the 16S and 23S rRNA genes of a blue-green alga: strong homology to chloroplast tRNA genes and tRNA genes of E. coli rrnD cluster.Nucleic Acids Res 11, 225-235.[Abstract]
Wilmotte, A. (1994). Molecular evolution and taxonomy of the cyanobacteria. In The Molecular Biology of Cyanobacteria, pp. 1-25. Edited by D. A. Bryant. The Netherlands: Kluwer Academic Publishers.
Wilmotte, A., Van der Auwera, G. & De Wachter, R. (1993). Structure of the 16S ribosomal RNA of the thermophilic cyanobacterium Chlorogloeopsis HTF (Mastigocladus laminosus HTF) strain PCC7518, and phylogenetic analysis.FEBS Lett 317, 96-100.[Medline]
Wilmotte, A., Neefs, J. M. & De Wachter, R. (1994). Evolutionary affiliation of the marine nitrogen-fixing cyanobacterium Trichodesmium sp strain NIBB 1067, derived by 16S ribosomal RNA sequence analysis.Microbiology 140, 2159-2164.[Abstract]
Wolk, C. P. (1996). Heterocyst formation.Annu Rev Genet 30, 59-78.[Medline]
Received 11 November 1999;
revised 24 January 2000;
accepted 13 March 2000.