School of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 1UG, UK1
Norwegian Institute for Water Research, PO Box 173 Kjelsas, N-0411 Oslo, Norway2
Author for correspondence: A. E. Walsby. Tel: +44 117 928 7490. e-mail: A. E.Walsby{at}bristol.ac.uk
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ABSTRACT |
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Keywords: cyanobacterial genetics, gas vesicles, Planktothrix
Abbreviations: 33RR, 33-residue repeat; pc, critical pressure; pt, turgor pressure; ph, hydrostatic pressure.
The GenBank accession numbers for the new sequences in this paper are AJ253125253133.
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INTRODUCTION |
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Gas vesicles are constructed from two principal proteins: GvpA is a small, highly conserved protein that forms the ribs of the cylindrical structure (Walker & Walsby, 1983 ; Hayes et al., 1986
); GvpC is a larger, less conserved protein, which is attached to the outer surface of the structure (Walsby & Hayes, 1988
) and stabilizes it (Hayes et al., 1992
). In Calothrix spp. there are two copies of gvpA, the gene that encodes the rib protein, located upstream of a single copy of gvpC, the gene that encodes the outer protein (Tandeau de Marsac et al., 1985
; Damerval et al., 1987
). The latter contains four partially conserved repeats of 99 nt, encoding four 33-residue repeats (33RRs) in the protein sequence. In Anabaena flos-aquae there are at least five copies of gvpA in tandem repeat (Hayes & Powell, 1995
) and one copy of gvpC, which contains five 99 nt elements (Hayes et al., 1988
). It has been suggested that the length of GvpC may influence the width of the gas vesicle as it assembles (Walsby, 1994
). In halobacteria, gas vesicle morphology is affected by mutations in gvpC (DasSarma et al., 1994
; Offner et al., 1996
). Changes in width will affect the critical pressure (pc) at which the gas vesicle collapses, and this has consequences for gas vesicles of organisms that occur in lakes that mix to different depths (Walsby, 1994
).
In Planktothrix spp., two forms of GvpC have been described, one of 16 kDa, encoded by gvpC16, and one of 20 kDa, encoded by gvpC20 (Beard et al., 1999 ). The larger GvpC possesses an additional sequence of 33 amino acids that shows similarity to three other 33-residue sections in the molecule. In strains of Planktothrix rubescens isolated from Lake Zürich, Switzerland, the gvpA and gvpC genes alternate in one of three arrangements: genotype GV1 contains gvpC20 only; GV2 contains gvpC20 and
C, an untranslated 72 bp fragment from the 3'-end of gvpC20; GV3 contains gvpC16, gvpC20 and
C (Beard et al., 1999
). Trichomes with genotype GV1 or GV2 produce gas vesicles that are wider and weaker (pc=0·861·0 MPa) than those with GV3 (pc=1·01·17 MPa) (Bright & Walsby, 1999
). These GV-genotypes can be distinguished by diagnostic PCR using primers complementary to sequences within gvpA or gvpC. This technique can be used to identify the genotype, and hence the pc phenotype, of single trichomes taken from a lake (Beard et al., 1999
).
Walsby et al. (1998) showed that during the winter months some Planktothrix trichomes in Lake Zürich are circulated to depths at which the hydrostatic pressure causes collapse of their gas vesicles. After cold winters, when the lake mixes to greater depths, this might lead to natural selection of strains with narrower and stronger gas vesicles, more of which remain buoyant and float upwards to form the spring inoculum. The proportions of strains with stronger gas vesicles can be investigated by PCR analysis of GV-genotypes.
It has been suggested that red strains of Planktothrix may be adapted to the conditions in deeper lakes. An investigation of seven strains revealed that four red-coloured strains possessed gas vesicles that collapsed at 0·91·0 MPa, while those in three green-coloured strains collapsed at a pressure of 0·60·7 MPa (Utkilen et al., 1985 ). We report here a survey of the gas-vesicle genotype of 71 strains of Planktothrix, isolated from 21 Nordic lakes ranging in depth from 8 to 67 m. In recent years these strains have been catalogued using the groupings suggested by Skulberg & Skulberg (1985)
, on the basis of pigmentation and trichome width: P. rubescens group, red forms that contain phycoerythrin (trichomes <6 µm, P. prolifica; >6 µm, P. rubescens); and P. agardhii group, green forms that lack phycoerythrin (trichomes <6 µm, P. agardhii; >6 µm, P. mougeotii). In this paper we retain the two group names for the different red and green strains, but do not distinguish them further. We report here the discovery, in many Planktothrix strains from Nordic lakes, of a longer variant of the gvpC gene that is correlated with the production of even wider and weaker gas vesicles. We investigated its association with the pigmentation of the strains, and its selection in relation to the depth of lakes in which they occur.
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METHODS |
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Isolation of genomic DNA.
Genomic DNA was isolated from Planktothrix cultures as described previously (Beard et al., 1999 ). Alternatively, the following procedure was used to prepare cell lysates that were also suitable templates for diagnostic PCR. A 1 ml sample from a dense culture (pressurized at 1·4 MPa to collapse the gas vesicles) was mixed with proteinase K (0·2 mg ml-1) and incubated for 15 min at 55 °C, followed by 15 min at 80 °C. After a brief centrifugation, the supernatant was collected, and 510 µl used in subsequent PCR amplifications. The cell lysates were stored at 4 °C for up to one year.
PCR amplification and sequencing.
Purified genomic DNA preparations or cell lysates were used as templates in PCR amplifications with the Planktothrix-specific gvp primers described in Table 1 and by Beard et al. (1999)
. All reactions were subjected to an initial denaturation step of 94 °C for 4 min, followed by 30 cycles of amplification (94 °C, 45 s; primer-specific annealing temperature, 1 min; 72 °C, 1 min), and a final extension step of 72 °C for 5 min. The annealing temperatures used for the diagnostic PCR amplifications were as follows: 65 °C (4 cycles) followed by 70 °C (26 cycles) for GVPA1 and GVPA2; 65 °C for GVPC1B, GVPC9 and GVPC11; 53 °C for GVP
4, GVP
5 and GVPC10. PCR products were purified and sequenced as described previously (Beard et al., 1999
).
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RESULTS |
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Arrangement and sequence of gas vesicle gene clusters
The arrangement and sequence of the gvpAC gene clusters in type strains of the three new GV genotypes were determined by the analysis of various PCR products (Fig. 2).
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Griffiths (1992) isolated from the gas vesicles of strain CYA 29 an SDS-soluble protein whose mobility in SDS-PAGE indicated a molecular mass of 28·8 kDa, close to the size calculated for the product of the gvpC28 gene. [The 21·7 kDa size for the product given by Griffiths et al. (1992)
was in error.]
Genotype GV6. P. agardhii CYA 137 was found to contain two consecutive copies of the gvpAgvpC28 repeat (Fig. 2). The sequence of the complete gvpC28 ORF shows two differences from that in P. rubescens PCC 7821. The sequences of two gvpAs and intergenic regions are identical to those in PCC 7821, except for one substitution in the gvpCgvpA spacer. The partial sequence of the downstream copy of gvpC28 differs from the upstream copy in three positions (see below).
Sequence differences in the gvpC variants
The 99 nt section that distinguishes gvpC20 from gvpC16 encodes a 33 amino acid residue section that can be considered to be a cryptic 33RR as it aligns with three other 33-residue sections with which it shows some sequence similarity (Beard et al., 1999 ). The additional 219 nt section that distinguishes gvpC28 from gvpC20 encodes an additional section of 73 amino acid residues. We have looked for evidence of 33RRs within this sequence: the sequence ER--VA-Q---QL occurs both at the C-terminal end of this segment and 33 residues before the end. Elsewhere, there is little sequence similarity.
Apart from the additional 99 nt and 219 nt sections that distinguish the variants gvpC16, gvpC20 and gvpC28, these genes show only a few single-nucleotide differences. These differences are restricted to six positions, which occur in regions common to all three of the variants. Three of these differences affect the encoded amino acid, as follows: Arg or Thr at residue 125, and Pro or Ser at residues 223 and 228 (positions given with respect to the sequence of GvpC28).
Survey of GV-genotypes of other Planktothrix strains in the NIVA collection
In 69 of the 72 Planktothrix strains in the NIVA collection isolated from lakes in Nordic countries, the GV-genotypes were determined, using three sets of PCR amplifications.
(1) The diagnostic PCR for gvpC (Fig. 1) was used to distinguish between genotypes GV1/2, GV3, GV4 and GV5/6.
(2) Amplification with primers GVPA1 and GVPA2 was used to screen for the presence of the C region between adjacent copies of gvpA (Beard et al., 1999
). This PCR was used to distinguish between genotypes GV1 and GV2, and between GV5 and GV6.
(3) Subclasses of genotypes GV2, GV4 and GV5, which differ according to the location and number of copies of C, were identified by amplification with primers GVP
4, GVP
5 and GVPC10 (Table 1
). The forward and reverse primers, GVP
4 and GVP
5, respectively, are specific to the unique sequence immediately upstream of
C, and are complementary to one another at their 5' ends. These primers generated a 584 bp product by amplification between two copies of
C located either side of a copy of gvpA. A second reverse primer GVPC10 (complementary to a region within gvpC) was included in this PCR to amplify the region between
C (GVP
4) and the next gvpC downstream: an 800 bp product indicated the gene arrangement
CgvpAgvpC20, whereas a 1019 bp product indicated the arrangement
CgvpAgvpC28.
P. rubescens group. Of 33 red-coloured strains of Planktothrix, 17 were of genotype GV2, 15 were of genotype GV4 and one (strain CYA 87 from Lake Levrasjön) was of genotype GV5a (Table 2). In five of seven cases where there was more than one isolate from a lake, the genotypes differed: both GV2 and GV5 occurred in Lake Levrasjön; GV2 and GV4 occurred in Lakes Kolbotnvatnet, Ören and Steinsfjorden; and three subclasses of GV4 occurred in Lake Gjersjøen. There was a discrepancy in the genotype of different cultures of P. rubescens CYA 18 (deposited as strain PCC 7821 at the Institut Pasteur): strain PCC 7821 was of genotype GV4a, whereas strain CYA 18 (NIVA Collection) was of genotype GV4b.
P. agardhii group. Of 38 green-coloured strains of Planktothrix, 5 were of genotype GV2a, 23 were of genotype GV5 and 10 were of genotype GV6. Again, the genotypes differed in five of the seven cases where there was more than one isolate from a lake: both GV5 and GV6 occurred in Lakes Fröylandsvatnet, Gjersjøen and Steinsfjorden, and both subclasses of GV5 occurred in Lakes rungen, Helgetjernet, Gjersjøen and Steinsfjorden. There was a discrepancy in the genotype of P. agardhii CYA 29: the culture maintained at Bristol since 1981 was of genotype GV5b (and represented the type strain of genotype GV5), but a culture sampled recently from the NIVA Collection was of genotype GV6. These discrepancies (and those observed in different cultures of P. rubescens CYA 18) may indicate that genetic rearrangements had occurred, or that the original isolates contained trichomes of different genotype.
pc values of gas vesicles
pc values were determined for 39 strains, representing most of the species and GV-genotype combinations available from each of the different lakes (Table 3). The principal finding was that the mean pc was primarily associated with GV-genotype. The range of mean pc for the different genotypes was: GV2, 0·790·91 MPa; GV4, 0·760·88 MPa; GV5, 0·610·75 MPa; GV6, 0·630·72 MPa. The frequency distribution of mean pc in GV-genotypes revealed no significant difference between GV2 and GV4, or between GV5 and GV6 (Fig. 3
. However, the difference between the two groups (GV2/4 with a mean pc of 0·85 MPa and GV5/6 with a mean of 0·65 MPa) is highly significant (t=12·9, P<0·001).
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DISCUSSION |
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Correlations between pc and lake depth
Many of the lakes from which the Planktothrix strains had been isolated were formed in glacial basins. The smaller lakes occupy basins of the kettle type or those formed by stream action. The maximum depths are given in Table 3.
We have previously suggested that there has been natural selection in planktonic cyanobacteria for gas vesicles of sufficiently high pc to withstand the maximum combination of cell turgor pressure (pt) and hydrostatic pressure (ph) in different lakes (Walsby, 1994 ; Walsby et al., 1999
). The simplistic expectation is that the pc should be sufficient to withstand the maximum combination of pressures (pt+ph) but should not exceed this combination. During the summer P. agardhii tends to occur in the epilimnion and P. rubescens in the metalimnion of lakes, at depths not usually exceeding 20 m (where ph=0·20 MPa); during active photosynthesis the pt may reach 0·4 MPa, giving a combined pressure (ph+pt) of 0·6 MPa. The mean pc exceeds this value in all of the strains investigated (Table 3
). The first expectation is therefore fulfilled for this period.
During the autumn and spring, however, Nordic lakes are mixed to their greatest depth (most are dimictic and turn over before and after formation of ice cover); a proportion of the Planktothrix population is then circulated to the lake bottom. During this period pt is unlikely to exceed 0·2 MPa. Table 3 lists the depths of the lakes from which the different strains were collected and the maximum combination of (ph+pt), where ph is calculated from the greatest depth (9810 Pa m-1 at 4 °C) and pt is assumed to be 0·2 MPa. Again, the mean pc exceeds (pt+ph) for strains from all of the lakes except the P. agardhii strains in the 64 m deep Lake Gjersjøen and the 67 m deep Gulf of Finland. For these strains, trichomes circulating below a depth of 50 m will lose buoyancy; they will be lost to the bottom sediment and will not contribute to the spring inoculum. The selection of strains with stronger gas vesicles depends on the relative costs of their production and the benefits they provide in decreasing these losses (see Walsby et al., 1999
, for a more detailed discussion). Of particular interest here is the occurrence of strains whose gas vesicle genotype is atypical for their species but is correlated with lake depth: P. agardhii CYA 91 has genotype GV2, which confers the medium pc of 0·92 MPa, sufficient to withstand the (ph+pt) of 0·84 MPa at the greatest depth, 64 m, in Lake Mälaren. Of the other two strains of P. agardhii with genotype GV2, strain 127 (pc=0·85 MPa) occurs in the moderately deep Vesijärvi (40 m) and strain 88/1 (pc=0·81 MPa) in the shallower Lake Ören (25 m).
Concerning the second expectation, provision of pc that does not exceed requirements, it is first noted that Planktothrix strains of genotype GV3 with the highest pc values (the dominant form in the 136 m deep Lake Zürich) are absent from all of the Nordic lakes investigated. We suggest that the strains of genotype GV3 are restricted in occurrence to the deepest lakes, like Lake Zürich. Six of the Nordic lakes contained strains of P. rubescens with genotype GV2/4 that have gas vesicles whose pc exceeds that required during winter mixing by 0·4 MPa or more (Table 3). There is, though, one exception: P. rubescens CYA 87 has the atypical genotype GV5 and gas vesicles of lower pc; it occurs in the shallow Lake Levrasjøn (15 m). This exception shows that the association between the GV2/4 genotype and the red strains, although strong, is not obligatory. Possible reasons why red strains with the GV5/6 genotype have not been selected in other lakes include the following: (1) insufficient time has occurred for selection since development of the population in the lake; (2) the benefit in reducing costs is insufficient; (3) the GV5/6 genotype is linked with other characters that are counterselective in these lakes; (4) the turgor pressures might be higher than 0·2 MPa in the red forms during periods of deep circulation; (5) the strains in culture are not all representative of the population in the lakes (P. rubescens of genotypes GV2 and GV5 occur in Levrasjøn).
Several lakes of different depths were shown to contain Planktothrix strains of both the GV2/4 and GV5/6 genotypes. This suggests that there is competition between strains producing the two types of gas vesicles: there should be selection for strains of genotype GV2/4, producing the stronger gas vesicles, following mixing events beyond 60 m; and there may be enrichment for strains of genotype GV5/6, producing the more efficient weaker gas vesicles, during summer growth in the metalimnion or epilimnion. This can now be investigated quantitatively by using the diagnostic PCR described in this paper to identify the GV-genotype of individual Planktothrix trichomes in lakewater samples.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 17 January 2000;
revised 7 March 2000;
accepted 22 May 2000.