Structure of the genes encoding the rod-core linker polypeptides of Mastigocladus laminosus phycobilisomes and functional aspects of the phycobiliprotein/linker-polypeptide interactions.

The 3' portion of the cpc operon in Mastigocladus laminosus encloses the genes 5'-cpcF-cpcG1-cpcG2-cpcG3 3'. The three cpcG genes encode different phycocyanin-associated rod-core linker polypeptides of the phycobilisomes with predicted 279, 247 and 254 amino acids in length. The gene products CpcG show a high similarity at their N-terminal domains (190 amino acids) and an overall identity of 47-53% to one another. Each of the three CpcG polypeptides is highly related to one of the four CpcG gene products of Anabaena sp. PCC 7120 (66-81% identity). It is suggested that these pairs of rod-core linker polypeptides mediate the same specific type of phycocyanin----allophycocyanin interaction in the similar phycobilisomes of M. laminosus and Anabaena sp. PCC 7120. The similarity of the CpcG1, CpcG2 and CpcG3 polypeptides to the single CpcG rod-core linker polypeptide of Synechococcus sp. PCC 7002 (36-41% identity) is lower. The rod-core linker polypeptides are more distantly related to the rod linker polypeptides associated with phycocyanin or phycoerythrin. However, six conserved domains were identified within the N-terminal 190 amino acids of these linker proteins, which bear similar amino acid sequences, including highly conserved basic amino acids. A similar amino acid sequence but with conserved acidic amino acids can be found in the beta subunits of phycocyanin, phycoerythrin and phycoerythrocyanin, which is protruding into the central cavity of the phycobiliprotein hexamers. It is suggested that these domains are sites of phycobiliprotein-hexamer/rod and rod-core linker interactions.     

A small multigene family encodes the rod-core linker polypeptides of Anabaena sp. PCC7120 phycobilisomes.

The cpc operon of Anabaena sp. PCC7120 is shown to encode ten genes: 5'-cpcB-cpcA-cpcC-cpcD-cpcE-cpcF- cpcG1-cpcG2-cpcG3-cpcG4-3'. The 3' portion of this operon includes four tandemly repeated genes encoding phycocyanin (PC)-associated, rod-core linker polypeptides of the phycobilisomes (PBS). The products of these four genes are most similar at their N termini, and overall are 50-61% identical and 68-76% similar to one another. The four CpcG proteins of Anabaena sp. PCC7120 are 41-47% identical and 62-65% similar to the single CpcG rod-core linker protein in Synechococcus sp. PCC7002. The N-terminal domains of the polypeptides are also more distantly related to the conserved domains of other types of rod-linker polypeptides associated with PC, phycoerythrin, and allophycocyanin (AP). Three of these rod-core linker proteins (CpcG1, CpcG2, and CpcG4) were demonstrated to occur in isolated PBS by N-terminal amino acid sequence analyses. These results indicate that previously proposed models for the PBS of Anabaena sp. are incorrect. It is suggested that the PBS of Anabaena sp. have eight peripheral rods, each of which interacts with the AP of the core via a specific rod-core linker (CpcG) polypeptide.     

Characterization of phycobilisome glycoproteins in the cyanobacterium Anacystis nidulans R2.

Concanavalin A-reactive linker and anchor subunits of phycobilisomes from Anacystis nidulans R2 (H. C. Riethman, T. P. Mawhinney, and L. A. Sherman, FEBS Lett. 215:209-214, 1987) were purified electrophoretically and analyzed for carbohydrate composition and quantity. Different quantities of glucose and N-acetylgalactosamine were found on the concanavalin A-reactive subunits analyzed. Proteolytic analysis of the purified subunits suggested that small regions of the 33- and 27-kilodalton linker polypeptides previously shown to be important for in vitro phycobilisome assembly contained the concanavalin A-reactive carbohydrates present on these subunits. The linker and anchor subunits from the morphologically different phycobilisome of Synechocystis sp. strain PCC6714 were also shown to be concanavalin A reactive. Membranes from iron-starved Anacystis nidulans, which lack assembled phycobilisomes and are associated with glycogen deposits, were shown to be depleted of linker and anchor proteins and to accumulate very large quantities of a concanavalin A-reactive, extrinsic membrane glycoprotein. We suggest that this iron stress-induced glycoprotein is associated with the glycogen deposits on the thylakoid surface and that the glycosylation of phycobilisome linker and anchor subunits is involved in the physiological regulation of phycobilisome assembly and degradation.     

The rpaC gene product regulates phycobilisome-photosystem II interaction in cyanobacteria

State transitions in cyanobacteria are a physiological adaptation mechanism that changes the interaction of the phycobilisomes with the Photosystem I and Photosystem II core complexes. A random mutagenesis study in the cyanobacterium Synechocystis sp. PCC6803 identified a gene named rpaC which appeared to be specifically required for state transitions. rpaC is a conserved cyanobacterial gene which was tentatively suggested to code for a novel signal transduction factor. The predicted gene product is a 9-kDa integral membrane protein. We have further examined the role of rpaC by overexpressing the gene in Synechocystis 6803 and by inactivating the ortholog in a second cyanobacterium, Synechococcus sp. PCC7942. Unlike the Synechocystis 6803 null mutant, the Synechococcus 7942 null mutant is unable to segregate, indicating that the gene is essential for cell viability in this cyanobacterium. The Synechocystis 6803 overexpressor is also unable to segregate, indicating that the cells can only tolerate a limited gene copy number. The non-segregated Synechococcus 7942 mutant can perform state transitions but shows a perturbed phycobilisome-Photosystem II interaction. Based on these results, we propose that the rpaC gene product controls the stability of the phycobilisome-Photosystem II supercomplex, and is probably a structural component of the complex.     

聚球藻7942混养培养中碳代谢与能量利用

为了考察聚球藻7942在混养条件下的能量利用效率,分别以葡萄糖和乙酸为碳源开展了聚球藻7942的混养培养研究,并在此基础上利用代谢通量分析方法对聚球藻7942混养条件下的碳代谢和能量利用进行了探讨。结果表明:葡萄糖和乙酸均能促进藻细胞生长,且乙酸促进藻细胞生长的作用更为明显;葡萄糖利用可明显增加藻细胞糖酵解途径中碳代谢流量,而乙酸利用则导致糖酵解途径中碳代谢流量减小,两种有机碳源均增加了柠檬酸循环中碳代谢流量;有机碳源导致藻细胞光化学效率下降,而葡萄糖较之乙酸降低藻细胞光化学效率更为明显。虽然混养条件下光能的贡献率要小于光自养,但基于能量的细胞得率和能量转换率均高于光自养,光自养和以葡萄糖、乙酸为碳源的混养中基于ATP生成的能量转换效率分别为6.81%、7.43%和8.77%。     

Spectroscopic studies of cyanobacterial phycobilisomes lacking core polypeptides

Synechococcus sp. PCC 7002 (Agmenellum quadruplicatum PR6) genes encoding two highly conserved phycobilisome core polypeptides, a small linker polypeptide (LC8, apcC) and the allophycocyanin-B alpha-subunit (alpha APB, apcD), respectively, were interrupted by insertion of restriction fragments carrying the neomycin phosphotransferase gene of Tn5. The interrupted genes were used to transform Synechococcus sp. PCC 7002 to kanamycin resistance. The apcC- mutant assembled phycobilisomes lacking the LC8 polypeptide and the apcD- mutant assembled phycobilisomes lacking alpha APB. No other differences between the compositions of the mutant and wild-type phycobilisomes were detected. The apcC- strain grew about 25% more slowly than the wild-type, and its phycobilisomes dissociated more rapidly in 0.33 M Na/K-PO4 (pH 8.0) or in 0.75 M Na/K-PO4 at pH 8.0, at 40 degrees C, than did those of the wild-type. The phycobilisomes of this mutant were indistinguishable from those of the wild-type with respect to absorption and circular dichroism spectra, as well as time-resolved fluorescence emission. Steady-state emission spectra indicate a small decrease in long wavelength (680 nm) emission from the apcC- phycobilisomes and a complementary increase in shorter wavelength (665 nm) emission, relative to wild-type phycobilisomes. Strain apcD- phycobilisomes appear to be functionally indistinguishable from those of the wild-type, in spite of the absence of the two alpha APB subunits which bear terminal acceptor bilins. The only spectroscopic difference was seen in the steady-state fluorescence emission, for which the emission of the mutant was about 15% higher than that of the wild-type and was slightly blue-shifted. A phenotype has yet to be found for the apcD- mutation.     

General distribution of the nitrogen control gene ntcA in cyanobacteria.

The ntcA gene from Synechococcus sp. strain PCC 7942 encodes a regulatory protein which is required for the expression of all of the genes known to be subject to repression by ammonium in that cyanobacterium. Homologs to ntcA have now been cloned by hybridization from the cyanobacteria Synechocystis sp. strain PCC 6803 and Anabaena sp. strain PCC 7120. Sequence analysis has shown that these ntcA genes would encode polypeptides strongly similar (77 to 79% identity) to the Synechococcus NtcA protein. Sequences hybridizing to ntcA have been detected in the genomes of nine other cyanobacteria that were tested, including strains of the genera Anabaena, Calothrix, Fischerella, Nostoc, Pseudoanabaena, Synechococcus, and Synechocystis.     

Expression of two nblA-homologous genes is required for phycobilisome degradation in nitrogen-starved Synechocystis sp. PCC6803

One of the responses exhibited by cyanobacteria when they are limited for an essential nutrient is the rapid degradation of their light-harvesting complex, the phycobilisome. Phycobilisome degradation is an ordered proteolytic process, visible by a color change of the cyanobacterial cell from blue-green to yellow-green (chlorosis). The small polypeptide NblA plays a key role in degradation of phycobilisomes in Synechococcus sp. PCC7942. Unlike Synechococcus, Synechocystis sp. PCC6803 has two nblA-homologous genes, nblA1 and nblA2, which are contiguous on the genome. Here we show that nblA1 and nblA2 are simultaneously expressed in Synechocystis 6803 upon nitrogen deprivation, and are both required for phycobilisome degradation.     

Phycobiliprotein methylation. Effect of the gamma-N-methylasparagine residue on energy transfer in phycocyanin and the phycobilisome

The phycobiliproteins contain a conserved unique modified residue, gamma-N-methylasparagine at beta-72. This study examines the consequences of this methylation for the structure and function of phycocyanin and of phycobilisomes. An assay for the protein asparagine methylase activity was developed using [methyl-3H]S-adenosylmethionine and apophycocyanin purified from Escherichia coli containing the genes for the alpha and beta subunits of phycocyanin from Synechococcus sp. PCC 7002 as substrates. This assay permitted the partial purification, from Synechococcus sp. PCC 6301, of the activity that methylates phycocyanin and allophycocyanin completely at residue beta-72. Using the methylase assay, two independent nitrosoguanidine-induced mutants of Synechococcus sp. PCC 7942 were isolated that do not exhibit detectable phycobiliprotein methylase activity. These mutants, designated pcm 1 and pcm 2, produce phycocyanin and allophycocyanin unmethylated at beta-72. The phycobiliproteins in these mutants are assembled into phycobilisomes and can be methylated in vitro by the partially purified methylase from Synechococcus sp. PCC 6301. The mutants produce phycobiliproteins in amounts comparable to those of wild-type and the mutant and wild-type phycocyanins are equivalent with respect to thermal stability profiles. Monomeric phycocyanins purified from these strains show small spectral shifts that correlate with the level of methylation. Phycobilisomes from the mutant strains exhibit defects in energy transfer, both in vivo and in vitro, that are also correlated with deficiencies in methylation. Unmethylated or undermethylated phycobilisomes show greater emission from phycocyanin and allophycocyanin and lower fluorescence emission quantum yields than do fully methylated particles. The results support the conclusion that the site-specific methylation of phycobiliproteins contributes significantly to the efficiency of directional energy transfer in the phycobilisome.     

Functional characterization of the gene encoding UDP-glucose: tetrahydrobiopterin alpha-glucosyltransferase in Synechococcus sp. PCC 7942

In this study, we attempted to characterize the Synechococcus sp. PCC 7942 mutant resultant from a disruption in the gene encoding UDP-glucose: tetrahydrobiopterin alpha-glucosyltransferase (BGluT). 2D-PAGE followed by MALDI-TOF mass spectrometry revealed that phycocyanin rod linker protein 33K was one of the proteins expressed at lower level in the BGluT mutant. BGluT mutant cells were also determined to be more sensitive to high light stress. This is because photosynthetic O2 exchange rates were significantly decreased, due to the reduced number of functional PSIs relative to the wild type cells. These results suggested that, in Synechococcus sp. PCC 7942, BH4-glucoside might be involved in photosynthetic photoprotection.     

Phycobilisome Mobility in the Cyanobacterium Synechococcus sp. PCC7942 is Influenced by the Trimerisation of Photosystem I

We have constructed a mutant of the cyanobacterium Synechococcus sp. PCC7942 deficient in the Photosystem I subunit PsaL. As has been shown in other cyanobacteria, we find that Photosystem I is exclusively monomeric in the PsaL(-) mutant: no Photosystem I trimers can be isolated. The mutation does not significantly alter pigment composition, photosystem stoichiometry, or the steady-state light-harvesting properties of the cells. In agreement with a study in Synechococcus sp. PCC7002 [Schluchter et al. (1996) Photochem Photobiol 64: 53-66], we find that state transitions, a physiological adaptation of light-harvesting function, occur significantly faster in the PsaL(-) mutant than in the wild-type. To explore the reasons for this, we have used fluorescence recovery after photobleaching (FRAP) to measure the diffusion of phycobilisomes in vivo. We find that phycobilisomes diffuse, on average, nearly three times faster in the PsaL(-) mutant than in the wild-type. We discuss the implications for the mechanism of state transitions in cyanobacteria.     

Phycobilisome structure in the cyanobacteria Mastigocladus laminosus and Anabaena sp. PCC 7120.

Phycobilisomes of the cyanobacteria Mastigocladus laminosus and Anabaena sp. PCC7120 differ from typical tricylindrical, hemidiscoidal phycobilisomes in three respects. Firstly, size comparisons of the core-membrane linker phycobiliproteins (LCM) in different cyanobacteria by SDS/PAGE reveal an apparent molecular mass of 120 kDa for the LCM of M. laminosus and Anabaena sp. PCC7120. This observation suggests that the polypeptides of these species have four linker-repeat domains. Secondly, phycobilisomes of M. laminosus are shown to contain at least three, but most probably four, different rod-core linker polypeptides (LRC). These LRC, which attach the peripheral rods to the core and thereby make phycocyanin/allophycocyanin contacts, have been identified and characterized by N-terminal amino acid sequence analysis. Additionally, electron microscopy of phycobilisomes isolated from M. laminosus and Anabaena sp. PCC7120 reveals similar structures which differ from those of Calothrix sp. PCC7601 with their typical six, peripheral rods. Based upon protein-analytical results and a reinterpretation of the data of [Isono, T. & Katoh, T. (1987) Arch. Biochem. Biophys. 256, 317-324], we discuss structural implications of recent findings on the established hemidiscoidal model for the phycobilisomes of M. laminosus and Anabaena sp. PCC7120. Up to eight peripheral rods are suggested to radiate from a modified core substructure which contains two additional peripheral allophycocyanin hexamer equivalents that serve as the core-proximal discs for two peripheral rods.     

Molecular structure and enzymatic function of lycopene cyclase from the cyanobacterium Synechococcus sp strain PCC7942.

A gene encoding the enzyme lycopene cyclase in the cyanobacterium Synechococcus sp strain PCC7942 was mapped by genetic complementation, cloned, and sequenced. This gene, which we have named crtL, was expressed in strains of Escherichia coli that were genetically engineered to accumulate the carotenoid precursors lycopene, neurosporene, and zeta-carotene. The crtL gene product converts the acyclic hydrocarbon lycopene into the bicyclic beta-carotene, an essential component of the photosynthetic apparatus in oxygen-evolving organisms and a source of vitamin A in human and animal nutrition. The enzyme also converts neurosporene to the monocyclic beta-zeacarotene but does not cyclize zeta-carotene, indicating that desaturation of the 7-8 or 7'-8' carbon-carbon bond is required for cyclization. The bleaching herbicide 2-(4-methylphenoxy)triethylamine hydrochloride (MPTA) effectively inhibits both cyclization reactions. A mutation that confers resistance to MPTA in Synechococcus sp PCC7942 was identified as a point mutation in the promoter region of crtL. The deduced amino acid sequence of lycopene cyclase specifies a polypeptide of 411 amino acids with a molecular weight of 46,125 and a pI of 6.0. An amino acid sequence motif indicative of FAD utilization is located at the N terminus of the polypeptide. DNA gel blot hybridization analysis indicated a single copy of crtL in Synechococcus sp PCC7942. Other than the FAD binding motif, the predicted amino acid sequence of the cyanobacterial lycopene cyclase bears little resemblance to the two known lycopene cyclase enzymes from nonphotosynthetic bacteria. Preliminary results from DNA gel blot hybridization experiments suggest that, like two earlier genes in the pathway, the Synechococcus gene encoding lycopene cyclase is homologous to plant and algal genes encoding this enzyme.     

Isolation and characterization of a sulfur-regulated gene encoding a periplasmically localized protein with sequence similarity to rhodanese.

During sulfur-limited growth, the cyanobacterium Synechococcus sp. strain PCC 7942 loses most of its photosynthetic pigments and develops an increased capacity to acquire sulfate. Sulfur deprivation also triggers the synthesis of several soluble polypeptides. We have isolated a prominent polypeptide of 33 kDa that accumulates specifically under sulfur-limiting conditions. This polypeptide was localized to the periplasmic space. The gene for this protein (designated rhdA) was isolated and discovered to lie within a region of the Synechococcus sp. strain PCC 7942 genome that encodes components of the sulfate permease system. The mRNA for the 33-kDa protein accumulates to high levels within an hour after the cells are deprived of sulfur and drops rapidly when sulfur is added back to the cultures. The amino acid sequence of the protein has similarity to bovine liver rhodanese, an enzyme that transfers the thiol group of thiosulfate to a thiophilic acceptor molecule, and a rhodaneselike protein of Saccharopolyspora erythraea. A strain in which rhdA was interrupted by a drug resistance marker exhibited marginally lower levels of rhodanese activity but was still capable of efficiently utilizing a variety of inorganic sulfur sources. The possible role of this protein in the transport of specific sulfur compounds is discussed.     

Diffusion of phycobilisomes on the thylakoid membranes of the cyanobacterium Synechococcus 7942. Effects of phycobilisome size, temperature, and membrane lipid composition

A variant of fluorescence recovery after photobleaching allows us to observe the diffusion of photosynthetic complexes in cyanobacterial thylakoid membranes in vivo. The unicellular cyanobacterium Synechococcus sp. PCC7942 is a wonderful model organism for fluorescence recovery after photobleaching, because it has a favorable membrane geometry and is well characterized and transformable. In Synechococcus 7942 (as in other cyanobacteria) we find that photosystem II is immobile, but phycobilisomes diffuse rapidly on the membrane surface. The diffusion coefficient is 3 x 10(-10) cm(2) s(-1) at 30 degrees C. This shows that the association of phycobilisomes with reaction centers is dynamic; there are no stable phycobilisome-reaction center complexes in vivo. We report the effects of mutations that change the phycobilisome size and membrane lipid composition. 1) In a mutant with no phycobilisome rods, the phycobilisomes remain mobile with a slightly faster diffusion coefficient. This confirms that the diffusion we observe is of intact phycobilisomes rather than detached rod elements. The faster diffusion coefficient in the mutant indicates that the rate of diffusion is partly determined by the phycobilisome size. 2) The temperature dependence of the phycobilisome diffusion coefficient indicates that the phycobilisomes have no integral membrane domain. It is likely that association with the membrane is mediated by multiple weak interactions with lipid head groups. 3) Changing the lipid composition of the thylakoid membrane has a dramatic effect on phycobilisome mobility. The results cannot be explained in terms of changes in the fluidity of the membrane; they suggest that lipids play a role in controlling phycobilisome-reaction center interaction.     

Changes in polypeptide composition of Synechocystis sp. strain 6308 phycobilisomes induced by nitrogen starvation.

Phycobilisomes isolated from actively growing Synechocystis sp. strain 6308 (ATCC 27150) consist of 12 polypeptides ranging in molecular mass from 11.5 to 95 kilodaltons. The phycobilisome anchor and linker polypeptides are glycosylated. Nitrogen starvation causes the progressive loss of phycocyanin and allophycocyanin subunits with molecular masses between 16 and 20 kilodaltons and of two linker polypeptides with molecular masses of 27 and 33 kilodaltons. Nitrogen starvation also leads to enrichment of four additional polypeptides with molecular masses of 46, 53, 57, and 61 kilodaltons and a transient enrichment of 35- and 41-kilodalton polypeptides in isolated phycobilisomes. The 57-kilodalton additional polypeptide was identified by immunoblotting as the large subunit of ribulosebisphosphate carboxylase/oxygenase. Proteins with the same molecular weights as the additional polypeptides were also coisolated with the 12 phycobilisome polypeptides in the supernatant of nitrogen-replete Synechocystis thylakoid membranes extracted in high-ionic-strength buffer and washed with deionized water. These observations suggest that the additional polypeptides in phycobilisomes from nitrogen-starved cells may be soluble or loosely bound membrane proteins which associate with phycobilisomes. The composition and degree of association of phycobilisomes with soluble and adjacent membrane polypeptides appear to be highly dynamic and specifically regulated by nitrogen availability. Possible mechanisms for variation in the strength of association between phycobilisomes and other polypeptides are suggested.     

Molecular characterization and redox regulation of phosphoribulokinase from the cyanobacterium Synechococcus sp. PCC 7942

We isolated and characterized a gene encoding phosphoribulokinase (PRK) from Synechococcus sp. PCC 7942. The isolated sequence consisted of a 999 bp open reading frame encoding 333 amino acid residues of PRK. The PRK contained a pair of cysteinyl residues corresponding to Cys16 and Cys55 of spinach PRK regulated by a ferredoxin-thioredoxin system. However, there were seventeen amino acid residues lacking between the two cysteinyl residues compared with those of the chloroplastic enzyme in higher plants. The recombinant PRK of Synechococcus sp. PCC 7942 accounted for about 6-13% of the total soluble protein in the Escherichia coli. The specific activity of the enzyme was 230 micro mol min(-1) (mg protein)(-1). The enzyme activity was completely inactivated by treatment with 5,5'-dithiobis (2-nitrobenzoic acid) (cysteinyl residue-specific oxidant) or was decreased by treatment with H(2)O(2), but was more tolerant to oxidation than that of chloroplast. The oxidized PRK was fully activated by treatment with excessive dithiothreitol. Furthermore, incubation with 3 mM ATP protected the oxidation of the enzyme by either 5,5'-dithiobis (2-nitrobenzoic acid) or H(2)O(2). These results suggest Synechococcus sp. PCC 7942 PRK can be regulated by reversible oxidation/reduction in vitro, but might be resistant to oxidative inactivation in vivo.     

Efficient gene transfer in Synechococcus sp. strains PCC 7942 and PCC 6301 by interspecies conjugation and chromosomal recombination.

We developed a versatile, efficient genetic transfer method for Synechococcus sp. strains PCC 7942 and PCC 6301 that exceeds natural transformation efficiencies by orders of magnitude. As a test case, we complemented a histidine auxotroph and identified a hisS homolog of PCC 7942 as the complementing gene.     

Cytochrome c-553 is not required for photosynthetic activity in the cyanobacterium Synechococcus.

In cyanobacteria, the water-soluble cytochrome c-553 functions as a mobile carrier of electrons between the membrane-bound cytochrome b6-f complex and P-700 reaction centers of Photosystem I. The structural gene for cytochrome c-553 (designated cytA) of the cyanobacterium Synechococcus sp. PCC 7942 was cloned, and the deduced amino acid sequence was shown to be similar to known cyanobacterial cytochrome c-553 proteins. A deletion mutant was constructed that had no detectable cytochrome c-553 based on spectral analyses and tetramethylbenzidine-hydrogen peroxide staining of proteins resolved by polyacrylamide gel electrophoresis. The mutant strain was not impaired in overall photosynthetic activity. However, this mutant exhibited a decreased efficiency of cytochrome f oxidation. These results indicate that cytochrome c-553 is not an absolute requirement for reducing Photosystem I reaction centers in Synechococcus sp. PCC 7942.     

The pacL gene of Synechococcus sp. strain PCC 7942 encodes a Ca(2+)-transporting ATPase.

An ATP-dependent Ca2+ uptake activity was identified in plasma membrane vesicles prepared from Synechococcus sp. strain PCC 7942. This activity was insensitive to agents which collapse pH gradients and membrane potentials but sensitive to vanadate, indicating that the activity is catalyzed by a P-type Ca(2+)-ATPase. A gene was cloned from Synechococcus sp. strain PCC 7942 by using a degenerate oligonucleotide based on a sequence conserved among P-type ATPases. This gene (pacL) encodes a product similar in structure to eukaryotic Ca(2+)-ATPases. We have shown that pacL encodes a Ca(2+)-ATPase by demonstrating that a strain in which pacL is disrupted has no Ca(2+)-ATPase activity associated with its plasma membrane. In addition, Ca(2+)-ATPase activity was restored to the delta pacL strain by introducing pacL into a second site in the Synechococcus sp. strain PCC 7942 chromosome.