Metabolic control of acclimation to nutrient deprivation dependent on polyphosphate synthesis.

Polyphosphate, an energy-rich polymer conserved in all kingdoms of life, is integral to many cellular stress responses, including nutrient deprivation, and yet, the mechanisms that underlie its biological roles are not well understood. In this work, we elucidate the physiological function of this polymer in the acclimation of the model alga Chlamydomonas reinhardtii to nutrient deprivation. Our data reveal that polyphosphate synthesis is vital to control cellular adenosine 5'-triphosphate homeostasis and maintain both respiratory and photosynthetic electron transport upon sulfur deprivation. Using both genetic and pharmacological approaches, we show that electron flow in the energy-generating organelles is essential to induce and sustain acclimation to sulfur deprivation at the transcriptional level. These previously unidentified links among polyphosphate synthesis, photosynthetic and respiratory electron flow, and the acclimation of cells to nutrient deprivation could unveil the mechanism by which polyphosphate helps organisms cope with a myriad of stress conditions in a fluctuating environment.

Biosynthetic pathways of two polypeptide subunits of the light-harvesting chlorophyll a/b protein complex.

We have used an in vitro reconstitution system, consisting of cell-free translation products and intact chloroplasts, to investigate the pathway from synthesis to assembly of two polypeptide subunits of the light-harvesting chlorophyll-protein complex. These polypeptides, designated 15 and 16, are integral components of the thylakoid membranes, but they are products of cytoplasmic protein synthesis. Double immunodiffusion experiments reveal that the two polypeptides share common antigenic determinants and therefore are structurally related. Nevertheless, they are synthesized in vitro from distinct mRNAs to yield separate precursors, p15 and p16, each of which is 4,000 to 5,000 daltons larger than its mature form. In contrast to the hydrophobic mature polypeptides, the precursors are soluble in aqueous solutions. Along with other cytoplasmically synthesized precursors, p15 and p16 are imported into purified intact chloroplasts by a post-translational mechanism. The imported precursors are processed to the mature membrane polypeptides which are recovered exclusively in the thylakoids. The newly imported polypeptides are assembled correctly in the thylakoid lipid bilayer and they bind chlorophylls. Thus, these soluble membrane polypeptide precursors must move from the cytoplasm through the two chloroplast envelope membranes, the stroma, and finally insert into the thylakoid membranes, where they assemble with chlorophyll to form the light-harvesting chlorophyll protein complex.

Similarity of a chromatic adaptation sensor to phytochrome and ethylene receptors.

Complementary chromatic adaptation in cyanobacteria acts through photoreceptors to control the biosynthesis of light-harvesting complexes. The mutant FdBk, which appears black, cannot chromatically adapt and may contain a lesion in the apparatus that senses light quality. The complementing gene identified here, rcaE, encodes a deduced protein in which the amino-terminal region resembles the chromophore attachment domain of phytochrome photoreceptors and regions of plant ethylene receptors; the carboxyl- terminal half is similar to the histidine kinase domain of two-component sensor kinases.

Cyanobacterial light-harvesting complex subunits encoded in two red light-induced transcripts.

The major light-harvesting complex in cyanobacteria and red algae, the phycobilisome, is composed of chromophoric and nonchromophoric polypeptides. Two linked genes encoding major chromophoric components, the polypeptide subunits of phycocyanin, were isolated from the cyanobacterium Fremyella diplosiphon. Transcripts from this phycocyanin subunit gene cluster were present as major species in the cyanobacterium grown in red light, but not in cultures maintained in green light. The genes for the subunits of the red light-induced phycocyanin were transcribed together (beta-phycocyanin followed by alpha-phycocyanin) on two messenger RNA species; one contained 1600 bases while the other had 3800 bases. The latter, which encompassed the smaller transcript, contained additional sequences extending from the 3' end of the coding region of the alpha-phycocyanin gene. It may encode other light-induced components of the phycobilisome. Since phycocyanin, which effectively absorbs red light, becomes a dominant constituent of the phycobilisome in red light, these different levels may reflect an important adaptive mechanism of these organisms to their environment.

Trophic conversion of an obligate photoautotrophic organism through metabolic engineering.

Most microalgae are obligate photoautotrophs and their growth is strictly dependent on the generation of photosynthetically derived energy. We show that the microalga Phaeodactylum tricornutum can be genetically engineered to thrive on exogenous glucose in the absence of light through the introduction of a gene encoding a glucose transporter (glut1 or hup1). This demonstrates that a fundamental change in the metabolism of an organism can be accomplished through the introduction of a single gene. This also represents progress toward the use of fermentation technology for large-scale commercial exploitation of algae by reducing limitations associated with light-dependent growth.

Mutants of Chlamydomonas with Aberrant Responses to Sulfur Deprivation.

In the absence of sulfur, Chlamydomonas reinhardtii, a unicellular green alga, increases its rate of sulfate import and synthesizes several periplasmic proteins, including an arylsulfatase (Ars). These changes appear to help cells acclimate to a sulfur-deficient environment. The elevated rate of sulfate import results from an increase in the capacity and affinity of the transport system for sulfate. The synthesis of Ars, a periplasmic enzyme that cleaves sulfate from aromatic compounds, enables cells to use these molecules as a source of sulfur when free sulfate is not available. To characterize the ways in which C. reinhardtii perceives changes in the sulfur status of the environment and regulates its responses to these changes, we mutagenized cells and isolated strains exhibiting aberrant accumulation of Ars activity. These mutants were characterized for Ars activity, ars mRNA accumulation, periplasmic protein accumulation, and sulfate transport activity when grown in both sulfur-sufficient and sulfur-deficient conditions. All of the mutants exhibited pleiotropic effects with respect to several of these responses. Strains harboring double mutant combinations were constructed and characterized for Ars activity and ars mRNA accumulation. From the mutant phenotypes, we inferred that both positive and negative regulatory elements were involved in the acclimation process. Both the epistatic relationships among the mutations and the effects of the lesions on the responses of C. reinhardtii to sulfur limitation distinguished these mutants from similar mutants in Neurospora crassa.

Chlamydomonas Xanthophyll Cycle Mutants Identified by Video Imaging of Chlorophyll Fluorescence Quenching.

The photosynthetic apparatus in plants is protected against oxidative damage by processes that dissipate excess absorbed light energy as heat within the light-harvesting complexes. This dissipation of excitation energy is measured as nonphotochemical quenching of chlorophyll fluorescence. Nonphotochemical quenching depends primarily on the [delta]pH that is generated by photosynthetic electron transport, and it is also correlated with the amounts of zeaxanthin and antheraxanthin that are formed from violaxanthin by the operation of the xanthophyll cycle. To perform a genetic dissection of nonphotochemical quenching, we have isolated npq mutants of Chlamydomonas by using a digital video-imaging system. In excessive light, the npq1 mutant is unable to convert violaxanthin to antheraxanthin and zeaxanthin; this reaction is catalyzed by violaxanthin de-epoxidase. The npq2 mutant appears to be defective in zeaxanthin epoxidase activity, because it accumulates zeaxanthin and completely lacks antheraxanthin and violaxanthin under all light conditions. Characterization of these mutants demonstrates that a component of nonphotochemical quenching that develops in vivo in Chlamydomonas depends on the accumulation of zeaxanthin and antheraxanthin via the xanthophyll cycle. However, observation of substantial, rapid, [delta]pH-dependent nonphotochemical quenching in the npq1 mutant demonstrates that the formation of zeaxanthin and antheraxanthin via violaxanthin de-epoxidase activity is not required for all [delta]pH-dependent nonphotochemical quenching in this alga. Furthermore, the xanthophyll cycle is not required for survival of Chlamydomonas in excessive light.

Sequences controlling transcription of the Chlamydomonas reinhardtii beta 2-tubulin gene after deflagellation and during the cell cycle.

In Chlamydomonas reinhardtii, transcripts from the beta 2-tubulin gene (tubB2), as well as those from other tubulin-encoding genes, accumulate immediately after flagellar excision as well as at a specific time in the cell cycle. Control of tubB2 transcript accumulation following deflagellation is regulated, at least partially, at the transcriptional level. We have fused the tubB2 promoter to the arylsulfatase (ars) reporter gene, introduced this construct into C. reinhardtii, and compared expression of the chimeric gene with that of the endogenous tubB2 gene. After flagellar excision, transcripts from the tubB2/ars chimeric gene accumulate with kinetics similar to those of transcripts from the endogenous tubB2 gene. The tubB2/ars transcripts also accumulate in a cell cycle-specific manner; however, chimeric transcripts are more abundant earlier in the cell cycle than the endogenous tubB2 transcripts. To elucidate transcriptional control of tubB2, we have mutated or removed sequences in the tubB2 promoter and examined the effect on transcription. The tubB2 promoter shares features with the promoters of other tubulin-encoding genes; these include a GC-rich region between the TATA box and the transcription initiation site and multiple copies of a 10-bp sequence motif that we call the tub box. The tubB2 gene contains seven tub box motifs. Changing the GC-rich region to an AT-rich region or removing three of the seven tub box motifs did not significantly affect transcription of the chimeric gene. However, removing four or five tub box motifs prevented increased transcription following deflagellation and diminished cell cycle-regulated transcription from the tubB2 promoter.

Chlamydomonas reinhardtii and photosynthesis: genetics to genomics.

Genetic and physiological features of the green alga Chlamydomonas reinhardtii have provided a useful model for elucidating the function, biogenesis and regulation of the photosynthetic apparatus. Combining these characteristics with newly developed molecular technologies for engineering Chlamydomonas and the promise of global analyses of nuclear and chloroplast gene expression will add a new perspective to views on photosynthetic function and regulation.

Highly expressed and alien genes of the Synechocystis genome.

Comparisons of codon frequencies of genes to several gene classes are used to characterize highly expressed and alien genes on the SYNECHOCYSTIS: PCC6803 genome. The primary gene classes include the ensemble of all genes (average gene), ribosomal protein (RP) genes, translation processing factors (TF) and genes encoding chaperone/degradation proteins (CH). A gene is predicted highly expressed (PHX) if its codon usage is close to that of the RP/TF/CH standards but strongly deviant from the average gene. Putative alien (PA) genes are those for which codon usage is significantly different from all four classes of gene standards. In SYNECHOCYSTIS:, 380 genes were identified as PHX. The genes with the highest predicted expression levels include many that encode proteins vital for photosynthesis. Nearly all of the genes of the RP/TF/CH gene classes are PHX. The principal glycolysis enzymes, which may also function in CO(2) fixation, are PHX, while none of the genes encoding TCA cycle enzymes are PHX. The PA genes are mostly of unknown function or encode transposases. Several PA genes encode polypeptides that function in lipopolysaccharide biosynthesis. Both PHX and PA genes often form significant clusters (operons). The proteins encoded by PHX and PA genes are described with respect to functional classifications, their organization in the genome and their stoichiometry in multi-subunit complexes.

Evolution of the phycobiliproteins.

Amino acid sequence alignments and phylogenetic analyses have been used to examine the relationships among 100 phycobiliprotein sequences. The alignments revealed a number of highly conserved amino acid residues that are involved in chromophore attachment and conformation, alpha-beta interactions and phycobilisome assembly. The phylogenetic analysis confirmed that the phycobiliprotein subfamilies, previously classified by their biochemical and spectroscopic properties, also formed coherent evolutionary groups. The alpha and beta subunits formed two distinct evolutionary lines that originate from a common ancestor. The pattern of divergence among the alpha subfamilies was identical to that of the beta subfamilies, strongly suggesting that the alpha and beta subunits of each phycobiliprotein type have coevolved. The phylogenetic data support a monophyletic separation of the eukaryotic sequences from the extant cyanobacterial sequences. The eukaryotic phycoerythrins appeared more closely related to the marine Synechococcus phycoerythrins than to the other cyanobacterial phycoerythrins. The cryptophyte phycobiliproteins formed a monophyletic group within the rhodophyte lineage. In conjunction with other phylogenetic markers, the analysis of additional phycobiliprotein sequences may help to further resolve the relationships among phycobiliprotein-containing organisms.

Characterization of Sulfate Transport in Chlamydomonas reinhardtii during Sulfur-Limited and Sulfur-Sufficient Growth.

We have characterized sulfate transport in the unicellular green alga Chlamydomonas reinhardtii during growth under sulfur-sufficient and sulfur-deficient conditions. Both the Vmax and the substrate concentration at which sulfate transport is half of the maximum velocity of the sulfate transport (K1/2) for uptake were altered in starved cells: the Vmax increased approximately 10-fold, and the K1/2 decreased approximately 7-fold. This suggests that sulfur-deprived C. reinhardtii cells synthesize a new, high-affinity sulfate transport system. This system accumulated rapidly; it was detected in cells within 1 h of sulfur deprivation and reached a maximum by 6 h. A second response to sulfur-limited growth, the production of arylsulfatase, was apparent only after 3 h of growth in sulfur-free medium. The enhancement of sulfate transport upon sulfur starvation was prevented by cycloheximide, but not by chloramphenicol, demonstrating that protein synthesis on 80S ribosomes was required for the development of the new, high-affinity system. The transport of sulfate into the cells occurred in both the light and the dark. Inhibition of ATP formation by the antibiotics carbonylcyanide m-chlorophenylhydrazone and gramicidin-S and inhibition of either F- or P-type ATPases by N,N-dicyclohexylcarbodiimide and vanadate completely abolished sulfate uptake. Furthermore, nigericin, a carboxylate ionophore that exchanges H+ for K+, inhibited transport in both the light and the dark. Finally, uptake in the dark was strongly inhibited by valinomycin. These results suggest that sulfate transport in C. reinhardtii is an energy-dependent process and that it may be driven by a proton gradient generated by a plasma membrane ATPase.

Hyperbaric oxygen in the treatment of burns.

This article presents the rationale and methods used to employ hyperbaric oxygen (HBO) treatment for patients with burns at the Sherman Oaks Community Hospital. It is based upon an expanded concept of burn injury formation and healing. We believe that new knowledge of oxygen transport and storage in the tissues in the presence of HBO will alter our understanding of the process of wound healing and the application of HBO in treatment.

Thermal injuries as a result of CO2 laser resurfacing.

CO2 laser resurfacing of the face for fine wrinkles has gained great popularity over a short period of time. The use of the CO2 laser has proven to be effective in reducing or eliminating fine wrinkles. This tool in the surgeon's armamentarium has been added to those of dermabrasion and chemical peel. The theoretical advantage of the use of the CO2 laser for resurfacing has been better accuracy and reportedly more control of the depth of penetration. The use of the CO2 laser has been welcomed by many cosmetic surgeons. Until now, there have been few reported cases of complications with the use of the CO2 laser. To many, this would sound too good to be true; unfortunately, that is the case. The CO2 laser is a high-energy machine that can indeed cause thermal injury. This thermal injury can result in deep burns to the skin and hypertrophic scarring. We feel this is more common than is currently being reported, and we share our experience as a burn and wound care referral service. During an 18-month period, 20 consecutive patients were referred to our practice who had received injuries from the CO2 laser resurfacing laser. We present here in this review a summary of those injuries. The CO2 resurfacing laser is a very effective tool for the treatment of fine wrinkles, but it is not without the potential for serious complications. We urge caution with the use of the laser and prompt recognition and treatment of thermal injury to the skin.

Psychological regression and marital status: determinants in psychiatric management of burn victims.

Major burn trauma is ordinarily associated with psychological regression, which regularly assumes either an immature, dependent (childlike), or primitive (animal-like) form. Also, the severely burned patient is exquisitely responsive, both constructively and destructively, to behavioral nuances in his or her "significant other," typically, the spouse. Two variables, type of regression and marital status, provide an empirically derived rationale for the psychiatric treatment of behavioral problems affecting patient management, including especially (1) pain-related behavior, (2) intrusive reexperiencing of the trauma, (3) depletion/despair phenomena, and (4) problems related to scarring. Results are more favorable when regression is of the dependent type rather than primitive type. Treatment is enhanced when the partner in a committed relationship is included in the treatment program.

Volitional collapse (loss of the will to live) in patients with burn injuries. Treatment strategy.

Volitional collapse, or loss of the will to live, remains one of the most vexing of the various system failures complicating serious illness or injury. It resembles, but is distinguishable from, major depression. Two features of volitional collapse may sometimes be turned to therapeutic advantage: it releases the patient from a struggle that may be dissipating already depleted energy reserves, and it transfers survival responsibility from one who has given up to others who have not. Preferred treatment involves carefully orchestrated initiatives from the patient's physician, closest friend, and spouse; their leverage derives from traditional sources, most importantly psychologic transference, bonding, and conjugal commitment. A burn unit provides an ideal environment in which to study the disorder and its response to treatment.

Auditory and neuropsychiatric behavior patterns after electrical injury.

Major electrical injury causes widespread tissue destruction. Slow and incomplete functional recovery after electrocution-type injury has led clinicians to suspect residual brain damage. One hundred and one consecutive patients who were admitted to the hospital because of electrical injury were studied. Forty-eight had electric-current injury. The other 53 had flash, contact, or arcing burns (electrical injury without passage of current). A primary study cohort of 16 patients with electric-current injury and 18 patients who had electrical injury without passage of current received specialized trauma-based psychiatric treatment, which was coordinated with serial auditory and neurologic studies. This strategy served to highlight discrepancies between preinjury and postinjury performance. Twelve of 16 patients with electric-current injury showed neurobehavioral (organic) dysfunction after 1 year, which implied brain damage; eight showed persistent auditory changes. Four of 18 patients who had electrical injury without passage of current met criteria for post-traumatic stress disorder after 1 year; none had neurobehavioral or auditory dysfunction. These findings indicate that patients with electric-current injury are at risk for permanent auditory dysfunction and brain damage, whereas those with other types of electrical burns are not.