Syntheses of {delta}-aminolevulinic acid and chlorophyll during chloroplast formation in Chlorella protothecoides

Greening cells of Chlorella prolothecoides were assayed foractivity of the in vivo synthesis of ALA, which was markedlydeveloped during light-induced greening. Effects of CH on thesyntheses of ALA and chlorophyll were also examined. The resultsstrongly suggested that a labile enzyme is involved in ALA synthesis,and that continuous formation of the enzyme is required forthe greening of cells. However, the prompt suppression of chlorophyllsynthesis when CH was added to rapidly greening cells was foundto be attributable not to the blockage of ALA synthesis butto the suppression of some later process(es) in the course ofchlorophyll synthesis, under the conditions used. The valueof the Hill coefficient for the CH inhibition of chlorophyllsynthesis as well as the CH concentration which caused 50% inhibitionremained unaltered whether it was measured when the ALA synthesisactivity was greatly inhibited by CH or when the activity wasonly slightly suppressed. (Received November 11, 1974; )     

Phosphatidylethanolamine synthesis in ethanolamine-responsive and -nonresponsive cells in culture

Mammalian cells can be classified into two types based upon whether or not they show growth response to ethanolamine (Etn) in culture. The content of phosphatidylethanolamine (PE) in phospholipid and incorporation of radioactive Etn into the cells were examined in the Etn-responsive and -nonresponsive cells in order to elucidate the mechanisms of growth stimulation by Etn. In all Etn-responsive cells tested, 5 microM Etn significantly altered the composition of cellular phospholipid compared to that grown without Etn, while Etn-nonresponsive cells had a similar phospholipid composition whether the growth medium contained Etn or not. Using two rat mammary carcinoma cell lines, 64-24 (responsive type) and 22-1 (nonresponsive type), further studies were carried out. In 64-24 cells there was a proportional increase in PE content as the dosage of Etn in the medium was increased. The increase in PE content leveled off at 10 microM. Further, the increase in PE content was correlated with increased rate of growth. In contrast, PE content or growth rate did not change at all in 22-1 cells. In 64-24 cells radioactive Etn (0.1-50 microM) was incorporated four- to five-fold more efficiently into phospholipid, and the aqueous pool of precursors of PE was ten times less as compared to 22-1 cells, indicating that Etn-responsive cells utilize Etn supplied in the medium to synthesize PE far more efficiently than Etn-nonresponsive cells. De novo synthesis of PE must not be sufficient to support optimum growth in Etn-responsive cells.     

The toxin from a ParDE toxin‐antitoxin system found in Pseudomonas aeruginosa offers protection to cells challenged with anti‐gyrase antibiotics

Toxin‐antitoxin systems are mediators of diverse activities in bacterial physiology. For the ParE‐type toxins, their reported role of gyrase inhibition utilized during plasmid‐segregation killing indicates they are toxic. However, their location throughout chromosomes leads to questions about function, including potential non‐toxic outcomes. The current study has characterized a ParDE system from the opportunistic human pathogen Pseudomonas aeruginosa (Pa). We identified a protective function for this ParE toxin, PaParE, against effects of quinolone and other antibiotics. However, higher concentrations of PaParE are themselves toxic to cells, indicating the phenotypic outcome can vary based on its concentration. Our assays confirmed PaParE inhibition of gyrase‐mediated supercoiling of DNA with an IC₅₀ value in the low micromolar range, a species‐specificity that resulted in more efficacious inhibition of Escherichia coli derived gyrase versus Pa gyrase, and overexpression in the absence of antitoxin yielded an expected filamentous morphology with multi‐foci nucleic acid material. Additional data revealed that the PaParE toxin is monomeric and interacts with dimeric PaParD antitoxin with a K_D in the lower picomolar range, yielding a heterotetramer. This work provides novel insights into chromosome‐encoded ParE function, whereby its expression can impart partial protection to cultures from selected antibiotics.