IMPACT OF IRON LIMITATION ON THE PHOTOSYNTHETIC APPARATUS OF THE DIATOM CHAETOCEROS MUELLERI (BACILLARIOPHYCEAE)

Iron starvation induced marked increases in flavodoxin abundance and decreases in light-saturated and light-limited photosynthesis rates in the diatom Chaetoceros muelleri. Consistent with the substitution of flavodoxin for ferredoxin as an early response to iron starvation, increases of flavodoxin abundance were observed before declines of cell division rate or chl a specific photosynthesis rates. Changes in the abundance of flavodoxin after the addition of iron to iron-starved cells indicated that flavodoxin was not actively degraded under iron-replete conditions. Greater declines in light-saturated oxygen evolution rates than dark oxygen consumption rates indicated that the mitochondrial electron transfer chain was not affected as greatly by iron starvation as the photosynthetic electron transfer chain. The carbon:nitrogen ratio was unaffected by iron starvation, suggesting that photosynthetic electron transfer was a primary target of iron starvation and that reductions in nitrate assimilation were due to energy limitation (the C:N ratio would be expected to rise under nitrogen-limited but energy-replete conditions). Parallel changes were observed in the maximum light-saturated photosynthesis rate and the light-limited initial slope of the photosynthesis-light curve during iron starvation and recovery. The lowest photosynthesis rates were observed in iron-starved cells and the highest values in iron-replete cells. The light saturation parameter, I_k, was not affected by iron starvation, nor was the chl-to-C ratio markedly reduced. These observations were consistent with iron starvation having a similar or greater effect on photochemical charge separation in PSII than on downstream electron transfer steps. Declines of the ratio of variable to maximum fluorescence in iron-starved cells were consistent with PSII being a primary target of iron starvation. The functional cross-section of PSII was affected only marginally (<20%) by iron starvation, with the largest values observed in iron-starved cells. The rate constant for electron transfer calculated from fast repetition rate fluorescence was found to covary with the light-saturated photosynthesis rate; it was lowest in the most severely starved cells.     

Shotgun proteome analysis of Bordetella pertussis reveals a distinct influence of iron availability on the bacterial metabolism,virulence, and defense response

One of the mechanisms involved in host immunity is the limitation of iron accessibility to pathogens, which in turn provokes the corresponding physiological adaptation of pathogens. This study reports a gel‐free nanoLC‐MS/MS‐based comparative proteome analysis of Bordetella pertussis grown under iron‐excess and iron‐depleted conditions. Out of the 926 proteins covered 98 displayed a shift in their abundance in response to low iron availability. Forty‐seven of them were found to be increased in level while 58 were found with decreased protein levels under iron starvation. In addition to proteins previously reported to be influenced by iron in B. pertussis, we observed changes in metabolic proteins involved in fatty acid utilization and poly‐hydroxybutyrate production. Additionally, many bacterial virulence factors regulated by the BvgAS two‐component system were found at decreased levels in response to iron limitation. These results, together with the increased production of proteins potentially involved in oxidative stress resistance, seem to indicate that iron starvation provokes changes in B. pertussis phenotype that might shape host–pathogen interaction.     

Physiological effects of the addition of n-dodecane as an oxygen vector during steady-state Bacillus licheniformis thermophillic fermentations perturbed by a starvation period or a glucose pulse

The effect of the presence of n-dodecane as a potential oxygen vector during oxygen-limited continuous cultures of a Bacillus strain was studied, under extreme nutrient supply conditions: glucose excess, limitation and starvation. The addition of n-dodecane to the aqueous phase of a mechanically agitated and aerated fermentation increased the k_La by up to 35%. The n-dodecane additions to Bacillus licheniformis cells during starvation (oxygen limitation with concomitant glucose starvation) caused a severe detrimental progressive change in cell physiological state with respect to cytoplasmic membrane polarisation and permeability which was mitigated against by alleviating either the oxygen limitation (by increasing the mean energy dissipation rate or by the addition of n-dodecane as an oxygen vector) or by alleviating the carbon limitation (by resuming the carbon feed or by the addition of a glucose pulse). Further that during periods of excess glucose (glucose pulse) a much higher k_La was required to prevent the onset of anaerobic mixed acid fermentation than could be provided by the addition of n-dodecane alone. n-Dodecane can be used to increase the k_La when added in sufficient quantities to the aqueous phase of a mechanically agitated and aerated bioreactor but the magnitude of this increase is process and vessel geometry specific.     

Juvenile hormone titres and metabolism during starvation-induced supernumerary larval moulting of the tobacco hornworm, Manduca sexta L.

When tobacco hornworm (manduca sexta) larvae are starved for 5 days immediately after ecdysis to the 5th instar, then fed normal diet, they undergo a supernumerary moult instead of metamorphosis. During starvation the titre of juvenile hormone in the haemolymph increased to a maximum of 3 ng juvenile hormone I equivalents/ml (determined by the black Manduca larval bioassay) on the fourth day of starvation, then began a decline which continued through the subsequent feeding period. The changes in juvenile hormone titre were not attributable to changes in haemolymph volume during starvation (only a 5% decrease) and subsequent feeding. During starvation the esterase activity of the haemolymph declined 4-fold with a 2-fold larger decrease in the DFP-insensitive, presumably juvenile hormone specific, esterase activity. Both the total and the juvenile hormone-specific esterase activity then increased as a function of larval weight during the subsequent feeding period. As growth was slow in the prolongedly starved larvae, sufficient juvenile hormone was present at the time of prothoracicotropic hormone (PTTH) and ecdysteroid release at the beginning of the fourth day of feeding to prevent metamorphosis.     

Depletion of Cellular Iron by Curcumin Leads to Alteration in Histone Acetylation and Degradation of Sml1p in Saccharomyces cerevisiae

Curcumin, a naturally occurring polyphenolic compound, is known to possess diverse pharmacological properties. There is a scarcity of literature documenting the exact mechanism by which curcumin modulates its biological effects. In the present study, we have used yeast as a model organism to dissect the mechanism underlying the action of curcumin. We found that the yeast mutants of histone proteins and chromatin modifying enzymes were sensitive to curcumin and further supplementation of iron resulted in reversal of the changes induced by curcumin. Additionally, treatment of curcumin caused the iron starvation induced expression of FET3, FRE1 genes. We also demonstrated that curcumin induces degradation of Sml1p, a ribonucleotide reductase inhibitor involved in regulating dNTPs production. The degradation of Sml1p was mediated through proteasome and vacuole dependent protein degradation pathways. Furthermore, curcumin exerts biological effect by altering global proteome profile without affecting chromatin architecture. These findings suggest that the medicinal properties of curcumin are largely contributed by its cumulative effect of iron starvation and epigenetic modifications.     

Delayed Turnover of Unphosphorylated Ssk1 during Carbon Stress Activates the Yeast Hog1 Map Kinase Pathway

In Saccharomyces cerevisiae, the Hog1 mitogen-activated protein kinase (MAPK) pathway coordinates the adaptation to osmotic stress and was recently reported to respond to acute changes in glucose levels. Similarly as in osmotic stress, glucose starvation leads to a transient accumulation of Hog1 in the nucleus. However, the kinetics and the mechanism of Hog1 activation are different for these stress conditions. During osmotic shock the activation of Hog1 can be transduced by either the Sho1 or the Sln1/Ypd1/Ssk1 branch. During glucose starvation the phosphorylation of Hog1 is slower and is completely dependent on Ssk1, but independent of Sho1. To characterize the mechanism of activation of Hog1 during carbon stress, we examined the turnover of Ssk1 protein levels upon glucose starvation in the presence of cycloheximide and monitored protein levels by western blotting. Our data demonstrate that unphosphorylated Ssk1 was quickly degraded during exponential growth and after osmotic stress but remained remarkably stable during glucose limitation. We conclude that glucose starvation induces a delay in the turnover of unphosphorylated Ssk1, which is sufficient to activate the Hog1 MAPK pathway. Although unphosphorylated Ssk1 is known to be degraded by the proteasome, its stabilization is apparently not due to changes in cellular localization or decrease in ubiquitination levels during glucose limitation.     

Characterization of the Starvation-Survival Response of Staphylococcus aureus

The starvation-survival response of Staphylococcus aureus as a result of glucose, amino acid, phosphate, or multiple-nutrient limitation was investigated. Glucose and multiple-nutrient limitation resulted in the loss of viability of about 99 to 99.9% of the population within 2 days. The remaining surviving cells developed increased survival potential, remaining viable for months. Amino acid or phosphate limitation did not lead to the development of a stable starvation-survival state, and cells became nonculturable within 7 days. For multiple-nutrient limitation, the development of the starvation-survival state was cell density dependent. Starvation survival was associated with a decrease in cell size and increase in resistance to acid shock and oxidative stress. There was no evidence for the formation of a viable but nonculturable state during starvation as demonstrated by flow cytometry. Long-term survival of cells was dependent on cell wall and protein biosynthesis. Analysis of [³⁵S]methionine incorporation and labelled proteins demonstrated that differential protein synthesis occurred deep into starvation.     

Some physiological and biochemical changes in marine eukaryotic red tide alga Heterosigma akashiwo during the alleviation from iron limitation

Some physiological and biochemical changes in the marine eukaryotic red tide alga Heterosigma akashiwo (Hada) were investigated during the alleviation from iron limitation. Chlorophyll a/carotenoid ratio increases as a result of iron alleviation. In vivo absorption spectra of iron-limited cells showed a chlorophyll (Chl) absorption peak at 630 nm, 2 nm blue-shifted from the normal position. Low-temperature fluorescence emission spectra of the cells have one prominent Chl emission peak at 685 nm. The cells showed a decrease in fluorescence yield from 685 nm band during alleviation from iron limitation. Low-temperature fluorescence excitation spectra and room-temperature fluorescence spectra indicated an efficient excitation energy transfer in the cells alleviated from iron limitation. Photosynthetic efficiency and carbohydrate content per cell increased after alleviation from iron limitation. Total protein decreased in iron-limited cells, while iron deficiency induced the appearance of specific soluble proteins (17 and 55 kDa).     

Hydroxamate Production as a High Affinity Iron Acquisition Mechanism in Paracoccidioides Spp

Iron is a micronutrient required by almost all living organisms, including fungi. Although this metal is abundant, its bioavailability is low either in aerobic environments or within mammalian hosts. As a consequence, pathogenic microorganisms evolved high affinity iron acquisition mechanisms which include the production and uptake of siderophores. Here we investigated the utilization of these molecules by species of the Paracoccidioides genus, the causative agents of a systemic mycosis. It was demonstrated that iron starvation induces the expression of Paracoccidioides ortholog genes for siderophore biosynthesis and transport. Reversed-phase HPLC analysis revealed that the fungus produces and secretes coprogen B, which generates dimerumic acid as a breakdown product. Ferricrocin and ferrichrome C were detected in Paracoccidioides as the intracellular produced siderophores. Moreover, the fungus is also able to grow in presence of siderophores as the only iron sources, demonstrating that beyond producing, Paracoccidioides is also able to utilize siderophores for growth, including the xenosiderophore ferrioxamine. Exposure to exogenous ferrioxamine and dimerumic acid increased fungus survival during co-cultivation with macrophages indicating that these molecules play a role during host-pathogen interaction. Furthermore, cross-feeding experiments revealed that Paracoccidioides siderophores promotes growth of Aspergillus nidulans strain unable to produce these iron chelators. Together, these data denote that synthesis and utilization of siderophores is a mechanism used by Paracoccidioides to surpass iron limitation. As iron paucity is found within the host, siderophore production may be related to fungus pathogenicity.     

Clp-dependent proteolysis down-regulates central metabolic pathways in glucose-starved Bacillus subtilis

Entry into stationary phase in Bacillus subtilis is linked not only to a redirection of the gene expression program but also to posttranslational events such as protein degradation. Using ³⁵S-labeled methionine pulse-chase labeling and two-dimensional polyacrylamide gel electrophoresis we monitored the intracellular proteolysis pattern during glucose starvation. Approximately 200 protein spots diminished in the wild-type cells during an 8-h time course. The degradation rate of at least 80 proteins was significantly reduced in clpP, clpC, and clpX mutant strains. Enzymes of amino acid and nucleotide metabolism were overrepresented among these Clp substrate candidates. Notably, several first-committed-step enzymes for biosynthesis of aromatic and branched-chain amino acids, cell wall precursors, purines, and pyrimidines appeared as putative Clp substrates. Radioimmunoprecipitation demonstrated GlmS, IlvB, PurF, and PyrB to be novel ClpCP targets. Our data imply that Clp proteases down-regulate central metabolic pathways upon entry into a nongrowing state and thus contribute to the adaptation to nutrient starvation. Proteins that are obviously nonfunctional, unprotected, or even “unemployed” seem to be recognized and proteolyzed by Clp proteases when the resources for growth become limited.     

Effects of Iron Limitation on the Degradation of Toluene by Pseudomonas Strains Carrying the TOL (pWWO) Plasmid

Most aerobic biodegradation pathways for hydrocarbons involve iron-containing oxygenases. In iron-limited environments, such as the rhizosphere, this may influence the rate of degradation of hydrocarbon pollutants. We investigated the effects of iron limitation on the degradation of toluene by Pseudomonas putida mt2 and the transconjugant rhizosphere bacterium P. putida WCS358(pWWO), both of which contain the pWWO (TOL) plasmid that harbors the genes for toluene degradation. The results of continuous-culture experiments showed that the activity of the upper-pathway toluene monooxygenase decreased but that the activity of benzyl alcohol dehydrogenase was not affected under iron-limited conditions. In contrast, the activities of three meta-pathway (lower-pathway) enzymes were all found to be reduced when iron concentrations were decreased. Additional experiments in which citrate was used as a growth substrate and the pathways were induced with the gratuitous inducer o-xylene showed that expression of the TOL genes increased the iron requirement in both strains. Growth yields were reduced and substrate affinities decreased under iron-limited conditions, suggesting that iron availability can be an important parameter in the oxidative breakdown of hydrocarbons.