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S S Golden

Publications and source records attributed to S S Golden.

At least 55 records · Page 3Linked to original sources
PubMed1992

Transcriptional and posttranscriptional components of psbA response to high light intensity in Synechococcus sp. strain PCC 7942.

The psbA genes, which encode the D1 protein of photosystem II, constitute a multigene family in the cyanobacterium Synechococcus sp. strain PCC 7942. Levels of messages from the three psbA genes change rapidly when cells are shifted from low-light to high-light conditions: the psbAI message level drops, whereas psbAII and psbAIII message levels increase dramatically. We examined the potential contributions of transcriptional and posttranscriptional processes in these high-light responses by subjecting cells that had been grown in a turbidostat at a standard light intensity (130 microeinsteins [microE] m-2 s-1) to either the same or a higher light intensity (500 microE m-2 s-1) in the presence or absence of rifampin. Northern (RNA blot) analysis of RNA isolated from cells subjected to high light showed that the increases in psbAII and psbAIII transcripts were blocked by rifampin. This suggests a transcriptional induction of these genes at high light intensities. Increased mRNA stability does not contribute to their accumulation in high-light conditions, since their half-life values did not increase relative to the half-lives measured at the standard light intensity. The rate of disappearance of the psbAI transcript in cells shifted to high light was diminished when either transcription or translation was blocked by rifampin or chloramphenicol, suggesting that accelerated degradation of the message requires de novo synthesis of a protein factor. When rifampin was added 10 min after the shift to high light intensity rather than before the shift, psbAI and psbAIII messages, but not the psbAII message, decayed at a faster rate. Susceptibility of the psbAIII transcript to the high-light-induced factor was also demonstrated by addition of chloramphenicol prior to the shaft to high light. psbAIII transcript levels went up more than twofold higher in chloramphenicol-treated cells than in untreated cells, whereas psbAII transcript levels were affected by the inhibitor. These experiments provide evidence that either new or increased synthesis of a degradation factor which affects a subset of Synechococcus transcripts occurs in cells subjected to high light intensity.

Adaptation, Biological↗
PubMed1991

Sequence analysis and phylogenetic reconstruction of the genes encoding the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase from the chlorophyll b-containing prokaryote Prochlorothrix hollandica.

Prochlorophytes similar to Prochloron sp. and Prochlorothrix hollandica have been suggested as possible progenitors of the plastids of green algae and land plants because they are prokaryotic organisms that possess chlorophyll b (chl b). We have sequenced the Prochlorothrix genes encoding the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase(rubisco), rbcL and rbcS, for comparison with those of other taxa to assess the phylogenetic relationship of this species. Length differences in the large subunit polypeptide among all sequences compared occur primarily at the amino terminus, where numerous short gaps are present, and at the carboxy terminus, where sequences of Alcaligenes eutrophus and non-chlorophyll b algae are several amino acids longer. Some domains in the small subunit polypeptide are conserved among all sequences analyzed, yet in other domains the sequences of different phylogenetic groups exhibit specific structural characteristics. Phylogenetic analyses of rbcL and rbcS using Wagner parsimony analysis of deduced amino acid sequences indicate that Prochlorothrix is more closely related to cyanobacteria than to the green plastid lineage. The molecular phylogenies suggest that plastids originated by at least three separate primary endosymbiotic events, i.e., once each leading to green algae and land plants, to red algae, and to Cyanophora paradoxa. The Prochlorothrix rubisco genes show a strong GC bias, with 68% of the third codon positions being G or C. Factors that may affect the GC content of different genomes are discussed.

Amino Acid Sequence↗
PubMed1991

Expression of the psbDII gene in Synechococcus sp. strain PCC 7942 requires sequences downstream of the transcription start site.

The psbDI and psbDII genes in Synechococcus sp. strain PCC 7942 encode the D2 polypeptide, an essential component of the photosystem II reaction center. Previous studies have demonstrated that transcripts from psbDII, but not psbDI, increase in response to high light intensity. Soluble proteins from Synechococcus cells shifted to high light were found to have affinity for DNA sequences upstream from the psbDII coding region. DNA mobility-shift and copper-phenanthroline footprinting assays of a 258-bp fragment revealed three distinct DNA-protein complexes that mapped to the untranslated leader region between +11 and +84. Deletion of the upstream flanking region to -42 had no effect on the expression of a psbDII-lacZ reporter gene or its induction by light, whereas a promoterless construct supported only minimal background levels of beta-galactosidase. A 4-bp deletion within the first protected region of the footprint decreased the beta-galactosidase activity to approximately 2% of that of the undeleted control, but gene expression remained responsive to light. Deletion of the three protected regions completely abolished both gene expression and light induction. These results suggest that the psbDII gene requires elements within the untranslated leader region for efficient gene expression, one of which may be involved in regulation by light.

Base Sequence↗
PubMed1990

Different and rapid responses of four cyanobacterial psbA transcripts to changes in light intensity.

The genome of the cyanobacterium Synechococcus sp. strain PCC 7942 contains three psbA genes which encode two forms of the D1 protein of photosystem II. Experiments using psbA-lacZ translational fusions and Western blot (immunoblot) analysis have shown that the psbA genes respond differently to changes in light intensity, altering the ratio of the two forms of D1 in the thylakoid membrane. Each gene produces a 1.2-kilobase (kb) mRNA. A probe specific for psbAII transcripts also identified a 1.6-kb mRNA which starts 419 base pairs upstream of the 5' end of the 1.2-kb species and overlaps the entire 1.2-kb transcript. This 419-base-pair region includes an open reading frame (ORF1) of 114 amino acids. We investigated the effects of changes in light intensity on psbAII transcript levels in a series of light shift experiments in the wild-type Synechococcus sp. and in AMC084, a mutant which does not produce the 1.6-kb transcript. After exposure to high light intensities for 15 min, the level of the 1.2-kb psbAII transcript increased in both strains. This transcript was not detected in either strain after transfer to low light intensity. The psbAIII transcript showed the same pattern of response as the 1.2-kb psbAII transcript, whereas the 1.6-kb psbAII transcript was unaffected by different light intensities. The psbAI transcript levels responded oppositely to those of psbAII and psbAIII. These data, considered along with previous results obtained by using lacZ translational gene fusions, indicate that the response of psbA genes to changes in light intensity is controlled primarily at the transcriptional level.

Amino Acid Sequence↗
PubMed1989

Light availability influences the ratio of two forms of D1 in cyanobacterial thylakoids.

The psbA multigene family in Synechococcus sp. strain PCC 7942 encodes two forms of the D1 protein; Form I, the product of psbAI, differs from Form II, the product of psbAII and psbAIII, at 25 of 360 amino acid positions. D1 is essential for photosynthesis as a protein component of the photosystem II reaction center. Antisera were raised against purified hybrid proteins encoded by psbAI-lacZ and psbAIII-lacZ translational gene fusions that contain the unique amino termini of Form I and Form II, respectively. Form specificity of each antiserum was verified by Western analysis using thylakoid membranes from mutant strains containing only Form I or Form II. Western analysis of thylakoid membranes from wild-type cells cultured at different light intensities detected both forms of D1 in the membrane and showed changes in the ratio of the two forms. The D1 composition of the membrane matched predicted ratios of the forms based on differential gene expression: psbAI is expressed highest at low light, and both psbAII and psbAIII are expressed highest at high light. Along a gradient of light intensity from 5 microE. m-2.s-1 to 482 microE.m-2.s-1, the relative amount of Form I in thylakoid membranes decreased 58%, while the relative amount of Form II increased 60%. Maximum detection of Form I coupled with minimum detection of Form II in membranes from cells harvested at light intensities below 390 microE.m-2.s-1 suggests a central role for Form I in photosystem II. Increased incorporation of Form II into the thylakoid membrane occurred at light intensities reported by others to be photoinhibitory, suggesting that Form II serves a role in adaptation to high light.

Chlorophyll↗
PubMed1989

psbA genes indicate common ancestry of prochlorophytes and chloroplasts.

It has long been suspected that chloroplasts evolved after an endosymbiotic event involving a photosynthetic prokaryote, presumably a cyanobacterium, and a eukaryotic organism. Recent studies have provided strong evidence about the cyanobacterial nature of chloroplasts. Since the discovery of prochlorophytes, oxygen-evolving photosynthetic prokaryotes containing chlorophyll a and chlorophyll b and lacking phycobiliproteins, there has been speculation that these represent evolutionary intermediates between cyanobacteria and chloroplasts. Prochloron sp., the first described prochlorophyte, proved difficult to work with because it is an obligate symbiont of marine ascidians. Prochlorothrix hollandica, a recently isolated, freshwater filamentous prochlorophyte, is easily maintained in the laboratory. Overall pigment composition and thylakoid membrane structure of P. hollandica suggest it has intermediate characteristics between cyanobacteria and the chloroplasts of higher plants. The P. hollandica psbA genes, which encode the photosystem II thylakoid protein D1, were cloned and sequenced and the sequences compared to those reported for cyanobacteria, a green alga, a liverwort, and several higher plants. The two psbA genes present in P. hollandica encode an identical amino-acid sequence. As in all chloroplast psbA genes, there is a seven amino-acid gap near the C terminus of the derived protein relative to the protein predicted by cyanobacterial genes, suggesting that P. hollandica is part of the lineage that led to chloroplasts after a divergence from cyanobacteria. This hypothesis is also supported by phylogenetic analysis of derived D1 amino-acid sequences from psbA genes of thirteen taxa on the basis of parsimony.

Amino Acid Sequence↗
PubMed1989

Genetic relationship of two highly studied Synechococcus strains designated Anacystis nidulans.

The cyanobacteria Synechococcus sp. strain PCC 7942 and Synechococcus sp. strain PCC 6301 are very closely related and both have been designated by the binomial Anacystis nidulans. The only established difference between the two strains is the superior transformation properties of strain PCC 7942. Significant homology between the rRNA genes of these strains was demonstrated by the ability of an rRNA operon from strain PCC 6301, interrupted by a spectinomycin and streptomycin resistance marker, to transform strain PCC 7942 by recombining with and replacing an endogenous rRNA operon. Restriction fragment length polymorphism data indicated that the chromosomes of the two strains were conserved around the three psbA loci, the two rRNA operons, and the psbDI locus. However, multiple polymorphisms were detected downstream of the psbDII locus, identifying a DNA rearrangement such as an inversion, insertion, or deletion within the chromosome. Analysis of genome structure by pulsed-field gel electrophoresis of large NotI restriction fragments showed only two bands that were visibly shifted between the chromosomes of the two strains. These data support their very close genetic relationship and the feasibility of studying genes derived from strain PCC 6301 in the highly transformable PCC 7942 strain.

Blotting, Southern↗
PubMed1989

Differential expression of members of a cyanobacterial psbA gene family in response to light.

The genome of the cyanobacterium Synechococcus sp. strain PCC 7942 contains three psbA genes encoding two forms of the D1 protein: form I, the product of psbAI, differs from form II, the product of both psbAII and psbAIII, at 25 of 360 residues. D1 is essential for photosynthesis as a core component of the photosystem II reaction center. Translational gene fusions between each of the Synechococcus psbA genes and the Escherichia coli lacZ gene were inserted into the chromosome of wild-type Synechococcus sp. at the respective psbA loci to serve as in vivo reporters of psbA expression. beta-Galactosidase activities indicated differential expression of the psbA-lacZ gene fusions related to light availability. Expression of psbAI was 500-fold greater than expression of psbAII and 50-fold greater than psbAIII under similar conditions. As light intensity decreased from 600 microE.m-2.s-1 to 2 microE.m-2.s-1, expression of the psbAI reporter increased eightfold while expression of the psbAII and psbAIII reporters decreased 10-fold, suggesting differential production of the two forms of D1 in photosystem II in response to light availability. Relative levels of psbA-lacZ fusion transcripts directly reflected beta-galactosidase activities in the transformants, although the fusion transcripts were less stable than native psbA messages.

Chlorophyll↗
PubMed1989

Two functional psbD genes in the cyanobacterium Synechococcus sp. strain PCC 7942.

The cyanobacterium Synechococcus sp. strain PCC 7942 has two copies of the psbD gene which encodes the D2 polypeptide of the photosystem II (PSII) reaction center. One of the genes, psbDI, overlaps the open reading frame of another photosystem II gene, psbC; the psbDII gene is monocistronic. Gene inactivation experiments had previously shown that psbDII is dispensable under normal laboratory growth conditions. However, similar experiments with psbDI never produced viable psbDI-inactivated mutants, presumably because psbC expression depends on transcription through psbDI. The experiments described here were designed to assess the need for psbDI independent of the need for expression of psbC. A strain, AMC027, was engineered in which a second copy of psbC was expressed from the psbDII locus. Northern (RNA) blot analysis confirmed that both psbDI and psbDII gave rise to dicistronic messages containing psbC sequences in AMC027. In this genetic background, it was possible to inactivate psbDI, creating strain AMC050 and indicating that the psbDII gene is functional. Western immunoblot analysis showed that the products of psbD and psbC, the PSII proteins D2 and CP43, respectively, were present in thylakoids of AMC050, but at reduced levels relative to the wild type, the mutant AMC027, and two psbDII-inactivated mutants. AMC050 consistently formed small colonies on plates and competed poorly in mixed-culture experiments. This suggested that, although not essential for viability, expression from the psbDI locus is required to produce sufficient D2 and CP43 for optimal growth.

Blotting, Southern↗
PubMed1988

Nucleotide sequence and transcript analysis of three photosystem II genes from the cyanobacterium Synechococcus sp. PCC7942.

The genome of the cyanobacterium Synechococcus sp. PCC7942 contains two genes encoding the D2 polypeptide of photosystem II (PSII), which are designated here as psbDI and psbDII. The psbDI gene, like the psbD gene of plant chloroplasts, is cotranscribed with and overlaps the open reading frame of the psbC gene, encoding the PSII protein CP43. The psbDII gene is not linked to psbC, and appears to be transcribed as a monocistronic message. The two psbD genes encode identical polypeptides of 352 amino acids, which are 86% conserved with the D2 polypeptide of spinach. In plants, the translational start codon of the psbC gene has been reported to be an ATG codon 50 bp upstream from the end of the psbD gene. This triplet is not present in the psbDI sequence of Synechococcus sp., but is replaced by ACG, a codon which is very unlikely to initiate translation. Translation of the psbC gene may begin at a GTG codon which overlap the psbDI open reading frame by 14 bp and is preceded by a block of homology to the 3' end of the 16S ribosomal RNA, a potential ribosome-binding site. There are only two bp differences between the sequences of the two psbD genes; one of these results in substitution in psbDII of GCG for the presumed GTG start codon in psbDI.

Amino Acid Sequence↗
PubMed1986

Expression of a family of psbA genes encoding a photosystem II polypeptide in the cyanobacterium Anacystis nidulans R2.

The genome of the cyanobacterium Anacystis nidulans R2 contains three genes (psbA) for the QB protein of photosystem II. This protein is essential for oxygenic photosynthetic electron tansport, and is the target for several herbicides which act by binding directly to the photosynthetic apparatus. Transcripts from the three Anacystis psbA genes are present in wild-type cells at different steady-state levels. The nucleotide sequences of two of the genes, psbAII and psbAIII, predict a protein having the same amino acid sequence which differs from that of the psbAI gene by 25 (out of 360) residues. Inactivation of each of the psbA genes in the Anacystis chromosome, singly or in pairs, shows that each of the genes is capable of producing sufficient functional QB protein to support normal photoautotrophic growth.

Amino Acid Sequence↗
PubMed1985

Mutation to herbicide resistance maps within the psbA gene of Anacystis nidulans R2.

A psbA gene encoding the target of photosystem II herbicide inhibition, the 32,000-dalton thylakoid membrane protein, has been cloned from a mutant of Anacystis nidulans R2, which is resistant to 3-(3,4-dichlorophenyl)-1,1-dimethylurea-(diuron). A cloned DNA fragment from within the coding region of this gene transforms wild-type cells to herbicide resistance, proving that mutation within psbA is responsible for that phenotype. The mutation consists of a single nucleotide change that replaces serine at position 264 of the wild-type protein with alanine in that of the diuron-resistant mutant.

Amino Acid Sequence↗
PubMed1984

Optimal conditions for genetic transformation of the cyanobacterium Anacystis nidulans R2.

Under optimal conditions, the cyanobacterium Anacystis nidulans R2 was transformed to ampicillin resistance at frequencies of greater than 10(7) transformants per microgram of plasmid (pCH1) donor DNA. No stringent period of competency was detected, and high frequencies of transformation were achieved with cultures at various growth stages. Transformation increased with time after addition of donor DNA up to 15 to 18 h. The peak of transformation efficiency (transformants/donor molecule) occurred at plasmid concentrations of 125 to 325 ng/ml with an ampicillin resistance donor plasmid (pCH1) and 300 to 625 ng/ml for chloramphenicol resistance conferred by plasmid pSG111. The efficiency of transformation was enhanced by excluding light during the incubation or by blocking photosynthesis with the electron transport inhibitor 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea (DCMU) or the uncoupler carbonyl cyanide-m-chlorophenyl hydrazone. Preincubation of cells in darkness for 15 to 18 h before addition of donor DNA significantly decreased transformation efficiency. Growth of cells in iron-deficient medium before transformation enhanced efficiency fourfold. These results were obtained with selection for ampicillin (pCH1 donor plasmid)- or chloramphenicol (pSG111 donor plasmid)-resistant transformants. Approximately 1,000 transformants per microgram were obtained when chromosomal DNA from an herbicide (DCMU)-resistant mutant was used as donor DNA. DCMU resistance was also transferred to recipient cells by using restriction fragments of chromosomal DNA from DCMU-resistant mutants. This procedure allowed size classes of fragments to be assayed for the presence of the DCMU resistance gene.

Ampicillin↗
PubMed1983

A hybrid plasmid is a stable cloning vector for the cyanobacterium Anacystis nidulans R2.

Anacystis nidulans R2 is a highly transformable strain which is suitable as a recipient for molecular cloning in cyanobacteria. In an effort to produce an appropriate cloning vector, we constructed a hybrid plasmid molecule, pSG111, which contained pBR328 from Escherichia coli and the native pUH24 plasmid of A. nidulans. pSG111 replicated in and conferred ampicillin and chloramphenicol resistance to both hosts. It contained unique sites for the restriction enzymes EcoRI, SalI, SphI, and XhoI, which could be used for the insertion of exogenous DNA. To demonstrate that a molecule like pSG111 could serve as a shuttle vector for the cloning of A. nidulans genes, we constructed a hybrid plasmid, pRNA404, containing an A. nidulans rRNA operon. This recombinant molecule was genetically and structurally stable during passage through A. nidulans and E. coli. The stability of the hybrid plasmid and the inserted rRNA operon demonstrates the feasibility of cloning in A. nidulans with hybrid vectors, with the subsequent retrieval of cloned sequences.

Cloning, Molecular↗