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.

Circadian rhythms in rapidly dividing cyanobacteria.

The long-standing supposition that the biological clock cannot function in cells that divide more rapidly than the circadian cycle was investigated. During exponential growth in which the generation time was 10 hours, the profile of bioluminescence from a reporter strain of the cyanobacterium Synechococcus (species PCC 7942) matched a model based on the assumption that cells proliferate exponentially and the bioluminescence of each cell oscillates in a cosine fashion. Some messenger RNAs showed a circadian rhythm in abundance during continuous exponential growth with a doubling time of 5 to 6 hours. Thus, the cyanobacterial circadian clock functions in cells that divide three or more times during one circadian cycle.

CikA, a bacteriophytochrome that resets the cyanobacterial circadian clock.

The circadian oscillator of the cyanobacterium Synechococcus elongatus, like those in eukaryotes, is entrained by environmental cues. Inactivation of the gene cikA (circadian input kinase) shortens the circadian period of gene expression rhythms in S. elongatus by approximately 2 hours, changes the phasing of a subset of rhythms, and nearly abolishes resetting of phase by a pulse of darkness. The CikA protein sequence reveals that it is a divergent bacteriophytochrome with characteristic histidine protein kinase motifs and a cryptic response regulator motif. CikA is likely a key component of a pathway that provides environmental input to the circadian oscillator in S. elongatus.

Circadian clock mutants of cyanobacteria.

A diverse set of circadian clock mutants was isolated in a cyanobacterial strain that carries a bacterial luciferase reporter gene attached to a clock-controlled promoter. Among 150,000 clones of chemically mutagenized bioluminescent cells, 12 mutants were isolated that exhibit a broad spectrum of periods (between 16 and 60 hours), and 5 mutants were found that show a variety of unusual patterns, including arrhythmia. These mutations appear to be clock-specific. Moreover, it was demonstrated that in this cyanobacterium it is possible to clone mutant genes by complementation, which provides a means to genetically dissect the circadian mechanism.

Expression of a gene cluster kaiABC as a circadian feedback process in cyanobacteria.

Cyanobacteria are the simplest organisms known to have a circadian clock. A circadian clock gene cluster kaiABC was cloned from the cyanobacterium Synechococcus. Nineteen clock mutations were mapped to the three kai genes. Promoter activities upstream of the kaiA and kaiB genes showed circadian rhythms of expression, and both kaiA and kaiBC messenger RNAs displayed circadian cycling. Inactivation of any single kai gene abolished these rhythms and reduced kaiBC-promoter activity. Continuous kaiC overexpression repressed the kaiBC promoter, whereas kaiA overexpression enhanced it. Temporal kaiC overexpression reset the phase of the rhythms. Thus, a negative feedback control of kaiC expression by KaiC generates a circadian oscillation in cyanobacteria, and KaiA sustains the oscillation by enhancing kaiC expression.

The cyanobacterial circadian system: a clock apart.

Several new molecular components of the circadian clocks of animals, fungi, and bacteria have been unveiled in the past two years. Enough parts are now identified to indicate that there is more than one way to build a biological clock, although there are parallels in the cycling molecular events among disparate groups of organisms.

Adaptive significance of circadian programs in cyanobacteria.

Prokaryotic cyanobacteria express robust circadian (daily) rhythms, even when growing with doubling times that are considerably faster than once every 24 h. This biological clock orchestrates cellular events to occur in an optimal temporal program. Competition experiments demonstrate that fitness is enhanced when the circadian period resonates with the period of the environmental cycle.

Nucleotide sequence of psbB from Synechococcus sp. strain PCC 7942.

The nucleotide sequence was determined for the Synechococcus sp. strain PCC 7942 psbB gene, which encodes the CP-47 protein of Photosystem II. The derived amino-acid sequence is highly conserved with those from other cyanobacterial and chloroplast psbB sequences. Transcript mapping experiments indicated two psbB transcription start sites in Synechococcus.

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.

Fasting in labor: relic or requirement.

OBJECTIVE: To evaluate the scientific literature on restrictions of eating and drinking during labor. DATA SOURCES: Computerized searches in MEDLINE and CINAHL, as well as historical articles, texts, and references cited in published works. Key words used in the searches included anesthesia in labor, childbirth, eating and drinking, epidural, fasting, fasting in labor, fasting and pregnancy, gastric aspiration, gastric emptying, intrapartum, intravenous fluids, i.v.s in labor, ketonuria, ketonuria in labor, parturition, pregnancy, and stomach contents in labor. STUDY SELECTION: Articles from indexed journals, excluding single-person case studies. DATA EXTRACTION: Data were extracted and organized under the following headings: historical review, effects of fasting on labor, research on maternal mortality/morbidity from aspiration, research on gastric emptying in labor, intravenous hydration in labor, and implications for nursing research. DATA SYNTHESIS: Research does not support restricting food and fluids in labor to prevent gastric aspiration. Restricting oral intake during labor has unexpected negative outcomes. CONCLUSIONS: Little is known about the differences in labor progress, birth outcomes, and neonatal status between mothers who consume food and/or fluids during labor and women who fast during labor. Research also is needed on the effects of epidural opioids on gastric emptying, nutritional requirements during labor, and the physiologic implications of fasting during labor. Fasting during labor is a tradition that continues with no evidence of improved outcomes for mother or newborn. Many facilities (especially birth centers) do not restrict eating and drinking. Across the United States, most hospitals restrict intake, usually to ice chips and sips of clear liquids. Anesthesia studies have focused on gastric emptying, measured by various techniques, presuming that delayed gastric emptying predisposes women to aspiration. Narcotic analgesia delays gastric emptying, but results are conflicting on the effect of normal labor and of epidural anesthesia on gastric emptying. The effect of fasting in labor on the fetus and newborn and on the course of labor has not been studied adequately. Only one study evaluated the probable risk of maternal aspiration mortality, which is approximately 7 in 10 million births.

Integrating the circadian oscillator into the life of the cyanobacterial cell.

In two decades, the study of circadian rhythms in cyanobacteria has gone from observations of phenomena in intractable species to the development of a model organism for mechanistic study, atomic-resolution structures of components, and reconstitution of a circadian biochemical oscillation in vitro. With sophisticated biochemical, biophysical, genetic, and genomic tools in place, the circadian clock of the unicellular cyanobacterium Synechococcus elongatus is poised to be the first for which a systems-level understanding can be achieved.

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.

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.

Adaptation to high light intensity in Synechococcus sp. strain PCC 7942: regulation of three psbA genes and two forms of the D1 protein.

The three psbA genes in the cyanobacterium Synechococcus sp. strain PCC 7942 encode two distinct forms of the D1 protein of photosystem II. The psbAI message, which encodes form I, dominates the psbA transcript pool at low to moderate light intensities; however, exposure to high light triggers a response in which the psbAI message is actively degraded while psbAII and psbAIII, which encode form II, are transcriptionally induced. We addressed whether these changes result from a generalized stress response and examined the consequence of light-responsive psbA regulation on the composition of D1 in thylakoid membranes. Heat shock and oxidative stress had some effect on levels of the three psbA transcripts but did not produce the responses generated by an increase in light intensity. Prolonged exposure to high light (24-h time course) was characterized by elevated levels of all psbA transcripts through maintenance of high levels of psbAII and psbAIII messages and a rebound of the psbAI transcript after its initial decline. Form II-encoding transcripts were enriched relative to those encoding form I at all high-light time points. Form II replaced form I in the thylakoid membrane at high light despite an abundance of psbAI transcript at later time points; this may be explained by the observed faster turnover of form I than form II in the membrane. We propose that form II is less susceptible to damage at high light and that this qualitative alteration, coupled with increased turnover of D1, protects the cells from photoinhibition.

mRNA stability is regulated by a coding-region element and the unique 5' untranslated leader sequences of the three Synechococcus psbA transcripts.

The psbAI and psbAIII transcripts in Synechococcus sp. strain PCC 7942 are subject to accelerated turnover when cells are exposed to high light intensities, but psbAII message stability is unaffected. We used a psbAI 'minigene' which has a part of the coding sequence removed as a reporter gene in order to identify the cis-acting elements of the transcript that determine stability. While engineering the minigene to optimally mimic the native gene, we identified a stabilizer element within the open reading frame, corresponding to the coding region for the first membrane span of the D1 protein, the presence of and translation through which was essential for normal psbA mRNA stability. We propose that this stabilizer is a site for ribosome pausing, and that accumulation of ribosomes on the transcript upstream of the pause site increases stability. To identify the elements that regulate the differential responses of the psbA transcripts to high-light growth, sequences from psbAII and psbAIII were substituted in the psbAI minigene reporter. The chimeric reporter transcripts established that the psbAI and psbAIII untranslated leaders determine the faster turnover of these messages. The untranslated leader regions of the psbA transcripts may regulate mRNA stability by modulating translation and thereby stability, or by recruiting RNA-binding proteins that affect mRNA turnover more directly.

Light-regulated expression of the psbD gene family in Synechococcus sp. strain PCC 7942: evidence for the role of duplicated psbD genes in cyanobacteria.

The genome of the cyanobacterium Synechococcus sp. strain PCC 7942 contains two psbD genes encoding the D2 protein of the photosystem II reaction center: psbDI, which is cotranscribed as a discistronic message with psbC (the gene encoding CP43, a chlorophyll-a binding protein), and psbDII, which is monocistronic. Northern blot analysis of psbD transcripts showed that the two genes responded differently when wild-type cells were shifted from moderate to high light intensity. Whereas psbDII transcripts increased 500% relative to unshifted control cells, psbDI-psbC transcripts remained unchanged. The beta-galactosidase activities expressed from translational fusions between the psbD genes and the Escherichia coli lacZ reporter gene displayed responses similar to those seen in the RNA. D2 protein levels in thylakoid membranes from wild-type cells increased to 250% of those of the unshifted control cells 12 h after a shift to high light intensities. In contrast, in a mutant strain (AMC016) that carries an inactive psbDII gene, D2 levels decreased by 50% under identical conditions. These results suggested that induction of psbDII gene expression by light can serve as a supplementary system for maintaining a functional photosystem II reaction center at high light intensity. This hypothesis was corroborated by mixed-culture experiments, in which AMC016 cells competed poorly with wild-type cells at high light intensity. These data suggest for the first time that differential expression of members of a cyanobacterial gene family serves to maintain a functional PSII reaction center under diverse environmental conditions.

Form II of D1 is important during transition from standard to high light intensity in Synechococcus sp. strain PCC 7942.

In Synechococcus sp. strain PCC 7942 the D1 protein of Photosystem II is encoded by a multigene family; psbAI encodes Form I of D1 whereas both psbAII and psbAIII encode Form II. The psbA genes are differentially regulated in response to changes in light intensity, such that psbAI expression and Form I predominate at standard light intensity, whereas psbAII and psbAIII are induced at high light intensity, causing insertion of Form II into the thylakoids. The present study addressed whether high-light induced Form II is important for Synechococcus cells during adaptation to high light intensity. Wild-type Synechococcus, and mutants which produce only Form I (R2S2C3) or only Form II (R2K1), were co-cultured at standard light (130 µE · m(-2) · s(-1)) and then shifted to high light (750 µE·m(-2)·s(-1)). Measurement of the proportion of each cell type at various time intervals revealed that the growth of R2S2C3, which has psbAII and psbAIII inactive, and thus lacks Form II, is transiently impaired upon shift to high light. Both mutants R2S2C3 and R2K1 maintained normal levels of psbA messages and D1 protein under standard and high light through an unknown mechanism that compensates for the inactive psbA genes. Thus, the impairment of R2S2C3 at high light is not due to a deficiency of D1 protein, but results from lack of Form II. We discounted the influence of possible secondary mutations by re-creating the psbA-inactivated mutants and testing the newly isolated strains. We conclude that Form II of D1 is intrinsically important for Synechococcus cells during a critical transition period after exposure to high light intensities.