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PubMed1992

The role of Lys272 in the pyridoxal 5-phosphate active site of Synechococcus glutamate-1-semialdehyde aminotransferase.

Glutamate-1-semialdehyde (GSA) aminotransferase catalyzes transfer of the C2 amino group of glutamate 1-semialdehyde to the C1 position to yield the tetrapyrrole precursor 5-aminolevulinate. Based on spectrophotometric and steady-state data, GSA aminotransferase is a B6-containing enzyme which uses a ping-pong bi-bi mechanism described for other aminotransferases. A putative active-site lysine at position 272 of Synechococcus GSA aminotransferase was replaced by Arg, Ile or Glu, and genes encoding the corresponding three site directed mutants were expressed in Escherichia coli. The catalytic competence of the resulting enzymes was determined. The similarity of the absorbance spectra of pyridoxal-P-treated forms of Lys272----Arg, Lys272----Ile, Lys272----Glu with free pyridoxal-P indicates that enzyme-bound pyridoxal-P does not form an internal aldimine in in these three site-directed mutants. Whereas Lys----Ile and Lys----Glu form only stable ketimines and aldimines with GSA and its analogues, addition of these compounds to the pyridoxamine-P and pyridoxal-P forms of Lys----Arg induces slow spectral changes, indicating the catalysis of a half-reaction with GSA, 4,5-dioxovalerate and 4,5-diaminovalerate. 5-Aminolevulinate apparently binds with both coenzyme forms of Lys272----Arg, however significant tautomeric rearrangement is only observed with the pyridoxal-P form. It is suggested that Lys272 is the covalent pyridoxal-P-binding site and that this catalytically active lysine residue channels the overall transamination reaction towards 5-aminolevulinate. The second-half reaction (4,5-diaminovalerate in equilibrium with 5-aminolevulinate) is possibly supported by the formation of an internal aldimine which correctly positions the C4 amino group of 4,5-diaminovalerate relative to the enzyme-bound pyridoxal-P.

Base Sequence↗

PubMed1996

Interaction between photosystem I and flavodoxin from the cyanobacterium Synechococcus sp. PCC 7002 as revealed by chemical cross-linking.

The interaction between photosystem I (PS I) and flavodoxin from the cyanobacterium Synechococcus sp. PCC 7002 was investigated by covalent cross-linking in the presence of a hydrophilic cross-linker, N- ethyl-3-(3-diaminopropyl)carbodiimide. Under the experimental conditions employed, five distinct cross-linking products of flavodoxin and PS I subunits are formed. Immunoblot analyses show that these species are the result of cross-linking of flavodoxin to PsaC, PsaD, an unidentified low-molecular-mass PS I polypeptide, and a 15-kDa subunit. The latter has been indirectly identified as the PsaF subunit. Analysis of the interaction of flavodoxin with PS I from a psaE mutant indicates that the PsaE subunit is required for correct complex formation between flavodoxin and PS I, although this subunit is not directly cross-linked to flavodoxin. In addition, the cross-linking products of PsaD with PsaC and PsaL, and PsaE with PsaF, are observed. The covalent complex of flavodoxin and PS I is shown to be fully inhibited with respect to electron transfer to soluble flavodoxin, ferredoxin or ferredoxin:NADP+ oxidoreductase.

Binding Sites↗

PubMed1995

A comparison of gene organization in the zwf region of the genomes of the cyanobacteria Synechococcus sp. PCC 7942 and Anabaena sp. PCC 7120.

The region of the genome encoding the glucose-6-phosphate dehydrogenase gene zwf was analysed in a unicellular cyanobacterium, Synechococcus sp. PCC 7942, and a filamentous, heterocystous cyanobacterium, Anabaena sp. PCC 7120. Comparison of cyanobacterial zwf sequences revealed the presence of two absolutely conserved cysteine residues which may be implicated in the light/dark control of enzyme activity. The presence in both strains of a gene fbp, encoding fructose-1,6-bisphosphatase, upstream from zwf strongly suggests that the oxidative pentose phosphate pathway in these organisms may function to completely oxidize glucose 6-phosphate to CO2. The amino acid sequence of fructose-1,6-bisphosphatase does not support the idea of its light activation by a thiol/disulfide exchange mechanism. In the case of Anabaena sp. PCC 7120, the tal gene, encoding transaldolase, lies between zwf and fbp.

Amino Acid Sequence↗

PubMed1996

Expression of genes involved in phycocyanin biosynthesis following recovery of Synechococcus PCC 6301 from nitrogen starvation, and the effect of gabaculine on cpcBa transcript levels.

The effect of the tetrapyrrole biosynthesis inhibitor gabaculine on the expression of specific genes involved in phycocyanin biosynthesis was investigated in cultures of Synechococcus PCC6301 in nitrogen chlorosis, and during recovery to nitrogen sufficiency. Patterns of transcription of the cpcBA (phycocyanin subunits), hemL (glutamate semialdehyde aminotransferase) and hemB (aminolaevulinate dehydratase) genes were visualised by Northern blotting and gene product formation for cpcBA, hemL and the gene for glu tRNA synthetase were monitored by Western blotting. Inhibition of phycobilin biosynthesis by gabaculine greatly decreased production of phycocyanin protein and of cpcBA transcript, indicating a tight coordination of apoprotein biosynthesis with chromophore supply at the level of transcription. Different patterns of response were observed with the other genes at the level of transcript formation or gene product synthesis.

Blotting, Northern↗

PubMed1996

Characterization of the genes encoding a phosphate-regulated two component sensory system in the marine cyanobacterium Synechococcus sp. WH7803.

An oligomer probe was designed to detect the presence of a putative phoB gene in the genome of the marine, phycoerythrin-containing cyanobacterium Synechococcus sp. WH7803. A 2.2 kb PstI fragment, identified using this probe, was cloned and the complete nucleotide sequence determined. The fragment contained two open reading frames encoding polypeptides which display all the sequence features expected of the response regulator and histidine protein kinase elements of a two component sensory system. Northern analysis confirmed that transcription of these genes was induced by phosphate limitation. On the basis of the sequence similarities and the regulation of their transcription by the availability of inorganic phosphate (Pi) these open reading frames were designated as phoB and phoR, respectively.

Amino Acid Sequence↗

PubMed1997

Low-temperature-induced desaturation of fatty acids and expression of desaturase genes in the cyanobacterium Synechococcus sp. PCC 7002.

Changes in response to temperature of lipid classes, fatty acid composition and mRNA levels for acyl-lipid desaturase genes were studied in the marine unicellular cyanobacterium, Synechococcus sp. PCC 7002. The degree of unsaturation of C18 fatty acids increased in cells grown at lower temperature for all lipid classes, and omega 3 desaturation occurred specifically in cells grown at low temperature. While the level of 18:1(9) fatty acids declined, desaturation at the omega 3 position of C18 fatty acids increased gradually during a 12-h period after a temperature shift-down to 22 degrees C. However, the mRNA levels of the desA (delta 12 desaturase), desB (omega 3 desaturase) and desC (delta 9 desaturase) genes increased within 15 min after a temperature shift-down to 22 degrees C; the desaturase gene mRNA levels also rapidly declined within 15 min after a temperature shift-up to 38 degrees C. Therefore, the elevation of mRNA levels for the desaturase genes is not the rate-limiting event for the increased desaturation of membrane lipids after a temperature shift-down. The rapid, low-temperature-induced changes in mRNA levels occurred even when cells were grown under light-limiting conditions for which the growth rates at 22 degrees C and 38 degrees C were identical. These studies indicate that the ambient growth temperature, and not some other growth rate-related process, regulates the expression of acyl lipid desaturation in this cyanobacterium.

Cold Temperature↗

PubMed1998

A mutant of Synechococcus PCC 7942 impaired in HCO3- uptake.

An inactivation library was used to isolate high-CO2-requiring mutants of Synechococcus PCC 7942. One of them, mutant IL-7, is composed of elongated cells, some 5-15 times longer than the wild-type. IL-7 is impaired in the ability to accumulate inorganic carbon within the cells due to a lesion in HCO3- transport. Consequently, the apparent photosynthetic affinity for external inorganic carbon was about 50-100-fold lower than in the wild-type. Analysis of the genomic region modified in IL-7 demonstrated that the inactivating fragment was composed of two genomically unrelated fragments which were ligated together during the formation of the inactivation library. One of the fragments originated from a known genomic region, rbcLS, encoding ribulose 1,5-bisphosphate carboxylase/oxygenase and the other showed high homology to mutS encoding a DNA mismatch repair protein. We suggest that the primary lesion in IL-7 was in mutS and not in rbcLS, and that the phenotype of IL-7 resulted from secondary random mutations. We were unable to identify the spontaneous mutation(s) due to low transformability of IL-7. Our finding that two unrelated fragments ligated together points to possible mistakes in the identification of the function of putative genes with the aid of an inactivation library.

Amino Acid Sequence↗

PubMed1998

Modification of topA in Synechococcus sp. PCC 7942 resulted in mutants capable of growing under low but not high concentration of CO2.

Insertion of a cartridge encoding kanamycin resistance within an open reading frame, ORF839, in the cyanobacterium Synechococcus sp. PCC 7942 resulted in merodiploids bearing both the normal and the modified ORF839, suggesting that its gene product is essential for growth. In the absence of kanamycin the mutants were able to grow like the wild type, but in its presence the mutants grew under 0.015% CO2 in air but not under 5% CO2 in air. ORF839, identified in this study, is highly homologous to topA encoding topoisomerase I in several organisms, but it does not contain the zinc-binding motif identified in the C-terminal region of the enzyme from Escherichia coli.

Carbon Dioxide↗

PubMed1999

Purification and characterization of cyanophycin and cyanophycin synthetase from the thermophilic Synechococcus sp. MA19.

The biosynthesis and accumulation of cyanophycin in the thermophilic cyanobacterium Synechococcus sp. MA19 were studied. By growing the cells in a 80-l closed tubular photobioreactor under controlled conditions, the cells accumulated cyanophycin amounting up to 3.5% of the dry cell matter. The cyanophycin was purified and chemical analysis showed that it was composed of arginine and aspartic acid occurring at a molar ratio of 1:0.9. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a broad distribution of the apparent molecular masses ranging from 20 to 130 kDa with a maximum at 50 kDa. During a three-step purification procedure involving ion exchange chromatography and gel filtration, the cyanophycin synthetase from strain MA19 was purified 144-fold to electrophoretic homogeneity. It consisted of only one single type of subunit exhibiting an apparent molecular mass of 130 kDa. The enzyme catalyzed the polymerization of arginine and aspartate at elevated temperatures and was even active at 80 degrees C.

Arginine↗

PubMed2000

The FAPY-DNA glycosylase (Fpg) is required for survival of the cyanobacterium Synechococcus elongatus under high light irradiance.

The gene for the DNA repair enzyme Fpg from Synechococcus elongatus was detected immediately downstream of the photosystem I gene psaE. fpg is likely expressed together with psaE by transcriptional readover while psaE is mostly expressed independently. Segregated psaE and fpg deletion strains were obtained upon insertional inactivation of both genes in S. elongatus. These mutants are viable under photoautotrophic conditions, but fail to grow under high light regimes that likely cause oxidative stress. These high light sensitive phenotypes suggest that the Fpg protein, which has been shown to repair DNA lesions caused by reactive oxygen species in Escherichia coli, may be involved in the photoprotection of cyanobacteria against oxidative damage caused under high irradiance.

Amino Acid Sequence↗

PubMed2002

Construction and initial characterization of a luminescent Synechococcus sp. PCC 7942 Fe-dependent bioreporter.

A Synechococcus sp. PCC 7942 bioreporter strain capable of sensing bioavailable Fe was constructed by fusing the Fe-responsive isiAB promoter to the Vibrio harveyi luxAB genes. Monitoring luxAB-dependent luminescence through the growth curve demonstrated that in Fe-replete media, transcription from the isiAB promoter was induced transiently in the mid-exponential phase of growth. The initiation of transcription was the functional response to a 10-fold depletion of intracellular Fe to approximately 12 amol Fe per cell. Constitutive isiAB-dependent transcription was observed in Fe-depleted growth media. A dose-response relationship of the bioreporter was generated using trace metal-buffered Fraquil medium and was best represented by a sigmoidal curve having a linear component extending between pFe 21.1 (Fe3+=10(-21.1) M) and pFe 20.6 (Fe3+)=10(-20.6) M). Initial field trials conducted using water sampled from Lake Erie demonstrate that the bioreporter can serve as a quantitative tool to assess Fe deficiency in natural freshwater environments.

Biosensing Techniques↗

PubMed1996

Characterization of psaI and psaL mutants of Synechococcus sp. strain PCC 7002: a new model for state transitions in cyanobacteria.

The psaI and psaL genes were characterized from the cyanobacterium Synechococcus sp. strain PCC 7002. The gene organization was different from that reported for other cyanobacteria with psaI occurring upstream and being divergently transcribed from the psaL gene. Mutants lacking PsaI or PsaL were generated by interposon mutagenesis and characterized physiologically and biochemically. Mutant strains PR6307 (delta psaI), PR6308 (psaI-) and PR6309 (psaL-) had doubling times similar to that of the wild type under both high- and low-intensity white light, but all grew more slowly than the wild type in green light. Only monomeric photosystem I (PS I) complexes could be isolated from each mutant strain when Triton X-100 was used to solubilize thylakoid membranes; however, approximately 10% of the PS I complexes from the psaI mutants, but not the psaL mutant, could be isolated as trimers when n-dodecyl beta-D-maltoside was used. Compositional analyses of the mutant PS I complexes indicate that the presence of PsaL is required for trimer formation or stabilization and that PsaI plays a role in stabilizing the binding of both PsaL and PsaM to the PS I complex. Strain PR6309 (psaL-) was capable of performing a state 2 to state 1 transition approximately three times more rapidly than the wild type. Because the monomeric PS I complexes of this mutant should be capable of diffusing more rapidly than trimeric complexes, these data suggest that PS I complexes rather than phycobilisomes might move during state transitions. A "mobile-PS I" model for state transitions that incorporates these ideas is discussed.

Amino Acid Sequence↗

PubMed1993

Isolation and Molecular Characterization of Five Marine Cyanophages Propagated on Synechococcus sp. Strain WH7803.

Five marine cyanophages propagated on Synechococcus sp. strain WH7803 were isolated from three different oceanographic provinces during the months of August and September 1992: coastal water from the Sargasso Sea, Bermuda; Woods Hole harbor, Woods Hole, Mass.; and coastal water from the English Channel, off Plymouth Sound, United Kingdom. The five cyanophage isolates were found to belong to two families, Myoviridae and Styloviridae, on the basis of their morphology observed in the transmission electron microscope. DNA purified from each of the cyanophage isolates was restricted with a selection of restriction endonucleases, and three distinguishably different patterns were observed. DNA isolated from Myoviridae isolates from Bermuda and the English Channel had highly related restriction patterns, as did DNA isolated from Styloviridae isolates from Bermuda and the English Channel. DNA isolated from the Myoviridae isolate from Woods Hole had a unique restriction pattern. The genome size for each of the Myoviridae isolates was ca. 80 to 85 kb, and it was ca. 90 to 100 kb for each of the Styloviridae isolates. Southern blotting analysis revealed that there was a limited degree of homology among all cyanophage DNAs probed, but clear differences were observed between cyanophage DNA from the Myoviridae and that from the Styloviridae isolates. Polypeptide analysis revealed a clear difference between Myoviridae and Styloviridae polypeptide profiles, although the major, presumably structural, protein in each case was ca. 53 to 54 kDa.

Journal Article↗

PubMed1994

Inactivation of ccmO in Synechococcus sp. Strain PCC 7942 Results in a Mutant Requiring High Levels of CO(2).

Inactivation of ccmO in Synechococcus sp. strain PCC 7942 resulted in a mutant which possesses aberrant carboxysomes and a normal inorganic carbon uptake capability but a reduced ability to photosynthetically utilize the internal inorganic carbon pool. Consequently, it exhibits low apparent photosynthetic affinity for extracellular inorganic carbon and demands high levels of CO(2) for growth.

Journal Article↗

PubMed1996

Enrichment culture and microscopy conceal diverse thermophilic Synechococcus populations in a single hot spring microbial mat habitat.

Recent molecular studies have shown a great disparity between naturally occurring and cultivated microorganisms. We investigated the basis for disparity by studying thermophilic unicellular cyanobacteria whose morphologic simplicity suggested that a single cosmopolitan species exists in hot spring microbial mats worldwide. We found that partial 16S rRNA sequences for all thermophilic Synechococcus culture collection strains from diverse habitats are identical. Through oligonucleotide probe analysis and cultivation, we provide evidence that this species is strongly selected for in laboratory culture to the exclusion of many more-predominant cyanobacterial species coexisting in the Octopus Spring mat in Yellowstone National Park. The phylogenetic diversity among Octopus Spring cyanobacteria is of similar magnitude to that exhibited by all cyanobacteria so far investigated. We obtained axenic isolates of two predominant cyanobacterial species by diluting inocula prior to enrichment. One isolate has a 16S rRNA sequence we have not yet detected by cloning. The other has a 16S rRNA sequence identical to a new cloned sequence we report herein. This is the first cultivated species whose 16S rRNA sequence has been detected in this mat system by cloning. We infer that biodiversity within this community is linked to guild structure.

Base Sequence↗

PubMed2001

Ecological physiology of Synechococcus sp. strain SH-94-5, a naturally occurring cyanobacterium deficient in nitrate assimilation.

Synechococcus sp. strain SH-94-5 is a nitrate assimilation-deficient cyanobacterium which was isolated from an ammonium-replete hot spring in central Oregon. While this clone could grow on ammonium and some forms of organic nitrogen as sole nitrogen sources, it could not grow on either nitrate or nitrite, even under conditions favoring passive diffusion. It was determined that this clone does not express functional nitrate reductase or nitrite reductase and that the lack of activity of either enzyme is not due to inactivation of the cyanobacterial nitrogen control protein NtcA. A few other naturally occurring cyanobacterial strains are also nitrate assimilation deficient, and phylogenetic analyses indicated that the ability to utilize nitrate has been independently lost at least four times during the evolutionary history of the cyanobacteria. This phenotype is associated with the presence of environmental ammonium, a negative regulator of nitrate assimilation gene expression, which may indicate that natural selection to maintain functional copies of nitrate assimilation genes has been relaxed in these habitats. These results suggest how the evolutionary fates of conditionally expressed genes might differ between environments and thereby effect ecological divergence and biogeographical structure in the microbial world.

Bacterial Proteins↗

PubMed2002

Molecular characterization of a thermostable cyanophycin synthetase from the thermophilic cyanobacterium Synechococcus sp. strain MA19 and in vitro synthesis of cyanophycin and related polyamides.

The thermophilic cyanobacterium Synechococcus sp. strain MA19 contained the structural genes for cyanophycin synthetase (cphA) and cyanophycinase (cphB), which were identified, cloned, and sequenced in this study. The translation products of cphA and cphB exhibited high levels of similarity to corresponding proteins of other cyanobacteria, such as Anabaena variabilis and Synechocystis sp. Recombinant cells of Escherichia coli harboring cphA colinear with lacPO accumulated cyanophycin that accounted for up to 25% (wt/wt) of the dry cell matter in the presence of isopropyl-beta-D-thiogalactopyranoside (IPTG). The cyanophycin synthetase was enriched 123-fold to electrophoretic homogeneity from the soluble fraction of the recombinant cells by anion-exchange chromatography, affinity chromatography, and gel filtration chromatography. The purified cyanophycin synthetase maintained the parental thermophilic character and was active even after prolonged incubation at 50 degrees C; in the presence of ectoine the enzyme retained 90% of its activity even after 2 h of incubation. The in vitro activity of the enzyme depended on ATP, primers, and both substrates, L-arginine and L-aspartic acid. In addition to native cyanophycin, the purified enzyme accepted a modified cyanophycin containing less arginine, alpha-arginyl aspartic acid dipeptide, and poly-alpha,beta-DL-aspartic acid as primers and also incorporated beta-hydroxyaspartic acid instead of L-aspartic acid or L-canavanine instead of L-arginine at a significant rate. The lack of specificity of this thermostable enzyme with respect to primers and substrates, the thermal stability of the enzyme, and the finding that the enzyme is suitable for in vitro production of cyanophycin make it an interesting candidate for biotechnological processes.

Amino Acid Sequence↗

PubMed2003

Nitrogen-regulated hypermutator strain of Synechococcus sp. for use in in vivo artificial evolution.

Artificially evolved variants of proteins with roles in photosynthesis may be selected most conveniently by using a photosynthetic organism, such as a cyanobacterium, whose growth depends on the function of the target protein. However, the limited transformation efficiency of even the most transformable cyanobacteria wastes much of the diversity of mutant libraries of genes produced in vitro, impairing the coverage of sequence space. This highlights the advantages of an in vivo approach for generating diversity in the selection organism itself. We constructed two different hypermutator strains of Synechococcus sp. strain PCC 7942 by insertionally inactivating or nutritionally repressing the DNA mismatch repair gene, mutS. Inactivation of mutS greatly increases the mutation rate of the cyanobacterium's genes, leading to an up-to-300-fold increase in the frequency of resistance to the antibiotics rifampin and spectinomycin. In order to control the rate of mutation and to limit cellular damage resulting from prolonged hypermutation, we placed the uninterrupted mutS gene in the cyanobacterial chromosome under the transcriptional control of the cyanobacterial nirA promoter, which is repressed in the presence of NH(4)(+) as an N source and derepressed in its absence. By removing or adding this substrate, hypermutation was activated or repressed as required. As expected, hypermutation caused by repression in PnirA-mutS transformants led to an accumulation of spectinomycin resistance mutations during growth.

Adenosine Triphosphatases↗