Studies on the near-UV effect on carotenogenesis in Verticillium agaricinum

Carotenoids identified in Verticillium agaricinum under near-UV were beta-, zeta-, and gamma-carotenes, neurosporene, torulene, neurosporaxanthin and one of its esters. Evidence supports the proposal that gamma-carotene, and not torulene, is the immediate precursor of neurosporaxanthin. It is also suggested that phytochrome may be involved in the high irradiance reactions (HIR) causing carotenoid synthesis in this fungus although there is no knowledge of how this is effected. Spores grown under near-UV conditions varied in size and shape from those grown in the dark. A new pigment (390, 420 nm) is also proposed as the photoreceptor for carotenogenesis in V. agaricinum.     

An action spectrum for photoinduced sporulation in the fungus Trichoderma viride

When a dark grown colony of Trichoderma viride is exposed towhite light for a short time, sporulation occurs only in thenarrow region of mycelia produced just prior to illumination.The action spectrum of this light effect was obtained for wavelengthsbelow 520 mµ using monochromatic irradiation of knownintensity having a band width of 10 mµ. The action spectrumshows 4 distinct peaks at 320, 380, 430 and 480 mµ. Thewavelengths at 320 and 380 mµ are most effective in photoinducedsporulation. The longer wavelengths, 430 and 480 mµ, areconsiderably less effective. The possibility that a carotenoid or a flavin compound may bea photoreceptor in this photoinduced sporulation is discussed. (Received January 4, 1969; )     

Inhibition of the Induced Formation of Tryptophanase in Escherichia coli by Near-Ultraviolet Radiation

Induced formation of tryptophanase in Escherichia coli B/r is temporarily inhibited by near-ultraviolet (UV) irradiation. The inhibition is greater when irradiation is at 5 C than when at room temperature. Hence, the inhibition is the result of a photochemical, rather than photoenzymatic, alteration of some cellular component. The action spectrum has a peak in the region of 334 nm and is similar to that for growth delay. However, inhibition of tryptophanase formation is more sensitive to near-UV irradiation than are growth, respiration, and the induced formation of beta-galactosidase. Thus, for tryptophanase the lack of formation cannot be due to general inhibition of metabolism. Pyridoxal phosphate absorbs in the near-UV region of the spectrum and is a cofactor for tryptophanase, but this enzyme in induced cells is not inactivated by near UV-radiations. An experiment in which toluene-treated suspensions from irradiated and unirradiated cells were mixed showed that irradiation does not cause the formation of an inhibitor of tryptophanase activity. The possibility remains that the absorption of radiant energy by pyridoxal phosphate interferes with the synthesis of tryptophanase.     

Red pigment in Bacillus megaterium spores.

Bacillus megaterium QM B1551 spores contained a unique red pigment in their membranes that was not found in other species. This red pigment, presumably a carotenoid, was synthesized about the time of dipicolinic acid synthesis during sporulation and was associated with the forespores. A yellow pigment was synthesized during sporulation in rich medium and was found in the mother cell compartment. Although the yellow pigment was also associated with spores, it could be removed by two different extraction procedures without impairing germination; it was absent when sporulation occurred in a minimal medium. Although the yellow pigment of the mother cell appeared to be dispensable, the red pigment may serve a more critical function, such as membrane stabilization.     

Red pigment in Bacillus megaterium spores

Bacillus megaterium QM B1551 spores contained a unique red pigment in their membranes that was not found in other species. This red pigment, presumably a carotenoid, was synthesized about the time of dipicolinic acid synthesis during sporulation and was associated with the forespores. A yellow pigment was synthesized during sporulation in rich medium and was found in the mother cell compartment. Although the yellow pigment was also associated with spores, it could be removed by two different extraction procedures without impairing germination; it was absent when sporulation occurred in a minimal medium. Although the yellow pigment of the mother cell appeared to be dispensable, the red pigment may serve a more critical function, such as membrane stabilization.     

The functional domain of adenylate cyclase associated with entry into meiosis in Saccharomyces cerevisiae.

Diploid yeast cells that carry a part of the CYR1 gene deficient in a region coding for the N-terminal domain of adenylate cyclase were growth arrested and accumulated unbudded cells after inoculation into complete medium or nitrogen-free medium, but produced many cells which had one or more buds after incubation in sporulation medium. The cells incubated in sporulation medium had abnormal spindles which were free from the spindle pole bodies, larger in size, or frequently distributed in cytoplasm. The levels of cyclic AMP in these cells did not decrease to the wild-type level after transfer to the sporulation medium and remained at a constant level. The results suggest that the N-terminal domain of adenylate cyclase is associated with the regulatory function for sporulation. The environmental signals for sporulation may be transferred to the adenylate cyclase system through a factor that negatively interacts with the N-terminal domain of this enzyme.     

Role of cloned carotenoid genes expressed in Escherichia coli in protecting against inactivation by near-UV light and specific phototoxic molecules.

Genes controlling carotenoid synthesis were cloned from Erwinia herbicola and expressed in an Escherichia coli strain. Carotenoids protect against high fluences of near-UV (NUV; 320 to 400 nm) but not against far-UV (200-300 nm). Protection of E. coli cells was not observed following treatment with either psoralen or 8-methoxypsoralen plus NUV. However, significant protection of cells producing carotenoids was observed with three photosensitizing molecules activated by NUV (alpha-terthienyl, harmine, and phenylheptatriyne) which are thought to have the membrane as an important lethal target. Protection of carotenoid-producing cells against inactivation was not observed with acridine orange plus visible light but was seen with toluidine blue O plus visible light.     

Induced chirality of the light-harvesting carotenoid salinixanthin and its interaction with the retinal of xanthorhodopsin

In xanthorhodopsin, a retinal protein-carotenoid complex of Salinibacter ruber, the carotenoid salinixanthin functions as a light-harvesting antenna in supplying additional excitation energy for retinal isomerization and proton transport. Another retinal protein, archaerhodopsin, has been shown to contain a carotenoid, bacterioruberin, but without an antenna function. We report here that the binding site confers a chiral geometry on salinixanthin in xanthorhodopsin and confirm that the same is true for bacterioruberin in archaerhodopsin. Cell membranes containing these rhodopsins exhibit CD spectra with sharp positive bands in the visible region where the carotenoids absorb, and in the case of xanthorhodopsin a negative band at 536 nm, as well as bands in the UV region. The carotenoid in ethanol has very weak optical activity in the visible region of the spectrum. Denaturation of the opsin upon deprotonation of the Schiff base at pH 12.5 eliminates the induced CD bands in both proteins. In one of these proteins, but not in the other, the carotenoid binding site depends entirely on the retinal. Hydrolysis of the retinal Schiff base of xanthorhodopsin with hydroxylamine eliminates the induced CD bands of salinixanthin. In contrast, hydrolysis of the Schiff base in archaerhodopsin does not abolish the CD bands of bacterioruberin. Thus, consistent with its antenna function, the carotenoid binding site interacts closely with the retinal only in xanthorhodopsin, and this interaction is the major source of the CD bands. In this protein, protonation of the counterion with a decrease in pH from 8 to 5 causes significant changes in the CD spectrum. The observed spectral features suggest that binding of salinixanthin in xanthorhodopsin involves the cyclohexenone ring of the carotenoid and its conformational heterogeneity is restricted.     

Induction of Sporulation During Synchronized Chromosome Replication in Bacillus subtilis

Synchronous chromosome replication was obtained in a culture of Bacillus subtilis temperature-sensitive mutants growing in a rich medium. At intervals during this replication, samples of cells were transferred to a poor medium to induce sporulation. The results show that the capacity of B. subtilis for induced sporulation reaches a peak about 15 min after chromosome replication has begun. This capacity then declines rapidly, but can be restored by initiating a new round of deoxyribonucleic acid replication. The possibility is discussed that sporulation can be induced only when the chromosome replication fork is passing through a stage 0 operon and that this may be located in the cysA-sul(R) region of the chromosome.     

MORPHOGENESIS IN VERTICILLIUM: A SELF-PRODUCED,DIFFUSIBLE MORPHOGENETIC FACTOR

A microsclerotial isolate of Verticillium albo-atrum produces a diffusible morphogenetic factor (DMF). At certain levels, DMF stimulates production of microsclerotia and melanin and inhibits hyphal elongation and sporulation. At higher concentrations it can inhibit production of microsclerotia and melanin as well as germination of Verticillium spores. Near-ultraviolet radiation (mostly 3200–4000 A, emission peak 3650 A), which inhibits production of microsclerotia and melanin, appears to act by suppressing synthesis of DMF. Once formed, DMF withstood 5 days’ exposure to near-UV. Dark-reared liquid cultures (shake or still) produced DMF while those reared under near-UV did not. DMF is active from pH 4 to pH 10, seems to be non-volatile, is dialyzable and water-soluble. In preliminary tests, it was insoluble in ether, ethanol and methylene dichloride. Acetone inactivated it. It may be 1 substance or a group of substances. We have developed assays for DMF in agar and liquid media employing the germination of Verticillium spores.     

Oxidative mechanisms of toxicity of low-intensity near-UV light in Salmonella typhimurium.

The exposure of Salmonella typhimurium to environmentally relevant near-UV light stress has been studied by the use of a low-intensity, broad-band light source. The exposure of cells to such a light source rapidly induced a growth delay; after continuous exposure for 3 to 4 h, cells began to die at a rapid rate. The oxidative defense regulon controlled by the oxyR gene was involved in protecting cells from being killed by near-UV light. This killing may be potentiated by the overexpression of near-UV-absorbing proteins. These results are consistent with near-UV toxicity involving the absorption of light by endogenous photosensitizers, leading to the production of active oxygen species. We have shown, however, that one such species, H2O2, is not a major photoproduct involved in killing by near-UV light. Strains lacking alkyl hydroperoxide reductase were more sensitive to near-UV light, indicating that such hydroperoxides may be photoproducts. Near-UV exposure induced sensitivity to high salt levels, indicating that membranes may be a target of near-UV toxicity and a possible source of alkyl hydroperoxides. The demonstration of the inactivation of the heme-containing protein catalase indicates that direct destruction of UV-absorbing macromolecules could be another factor in near-UV toxicity. Cells which have been exposed to near-UV light for long, but sublethal, periods of time (up to 4 h) can recover and resume growth if the UV exposure is stopped but become progressively more sensitive to further stresses, such as H2O2. This result indicates that cells gradually accumulated damage during near-UV exposure until toxic levels were reached.     

The effects of light on a circadian rhythm of conidiation in neurospora

The expression of a circadian rhythm of conidiation by timex, a strain of Neurospora crassa, is inhibited by growth in continuous white light. The action spectrum for this effect has a strong peak (with minor subpeaks) in the blue region of the visible spectrum, and a broad shoulder in the near ultraviolet. This action spectrum suggests that a carotenoid or flavin compound may be the photoreceptor, but does not allow one to determine conclusively whether the receptor is indeed a carotenoid, flavin, or some other unrelated pigment. Two lines of evidence suggest that a carotenoid is not the photoreceptor. First, the in vivo absorption spectrum of timex (representing the sum of the spectra of the individual pigments present, predominantly carotenoids) has peaks at wavelengths 10 to 20 mμ longer than those of the action spectrum peaks. Second, an albino-timex has normal photosensitivity, a situation requiring that the photoreceptor, if carotenoid, be a quantitatively minor constituent of the total carotenoid complement.

The magnitude and direction of phase-shift resulting from a standard dose of white light given at different times in the daily cycle of timex varies in the manner reported for other organisms. Additional phase-shift experiments have shown that there are no major transients in the attainment of a new equilibrium after a phase-shifting perturbation, and that 2 light reactions (rapidly and slowly saturating) may be involved in the phase-shift response.

     

Near-UV stress in Salmonella typhimurium: 4-thiouridine in tRNA, ppGpp, and ApppGpp as components of an adaptive response.

We have examined the role of 4-thiouridine in the responses of Salmonella typhimurium to near-UV irradiation. Mutants lacking 4-thiouridine (nuv) and mutants defective in the synthesis of ppGpp (guanosine 5'-diphosphate-3'-diphosphate) (relA) were found to be sensitive to killing by near-UV. Near-UV induced the synthesis of a set of proteins that were not induced in the nuv mutant. Some of these proteins were identified as oxidative defense proteins, and others were identified as ppGpp-inducible proteins. Over 100-fold increases in ApppGpp (adenosine 5', 5"'-triphosphoguanosine-3"'-diphosphate, the adenylylated form of ppGpp) were observed in wild-type cells after near-UV irradiation but not in the 4-thiouridine-deficient mutant. These data support a model in which ppGpp and ApppGpp, a dinucleotide proposed to be synthesized by tRNA-aminoacyl synthetases as a response to the cross-linking of 4-thiouridine in tRNA by near-UV, induce the synthesis of proteins necessary for resistance to near-UV irradiation.     

Sugar-induced change in near ultraviolet circular dichroism of α-crystallin

In order to study the effect of sugar molecules on the structure of α-crystallin, the protein from bovine lenses was isolated and treated with D-glucose and D-glucose-6-phosphate. Structural studies of the products were carried out using circular dichroism (CD) measurements. In the near-ultraviolet (UV) region, three positive and two negative CD bands are observed for α-crystallin. Upon incubation with the saccharides, the positive ellipticities of these near-UV CD bands increase greatly. The CD spectrum of α-crystallin in the far-UV region, which is typical of the β-conformation of a protein, does not change. The increase does not appear to be caused by glycosylation, or by sugar metabolites such as sorbitol or glycolytic products. When the concentration of sugar is higher than that has been used in this study, the protein tends to aggregate. The results strongly suggest that sugar, upon interaction with α-crystallin, induces a change in the tertiary structure of the protein while its secondary structure remains intact. The interaction of sugar molecules causing such change is weak, noncovalent, and temperature-dependent. The near-UV CD of α-crystallin appears to be a useful probe for the study of such structural changes, which may be significant in the pathogenesis of cataract.     

Analysis of the germination of spores of Bacillus subtilis with temperature sensitive spo mutations in the spoVA operon

A Bacillus subtilis strain with a base substitution in the ribosome-binding site of spoVAC was temperature sensitive (ts) in sporulation and spores prepared at the permissive temperature were ts in L-alanine-triggered germination, but not in germination with Ca2+-dipicolinic acid (DPA) or dodecylamine. Spores of a ts spo mutant with a missense mutation in the spoVAC coding region were not ts for germination with l-alanine, dodecylamine or Ca2+-DPA. These findings are discussed in light of the proposal that SpoVA proteins are involved not only in DPA uptake during sporulation, but also in DPA release during nutrient-mediated spore germination.     

Amino acids in the N-terminal region regulate the photocycle of photoactive yellow protein.

The spectroscopic properties of photoactive yellow protein (PYP) partially digested by chymotrypsin were studied. Chymotrypsin yielded three major products that were yellow but distinguishable by SDS-PAGE. They were readily separated by DEAE-Sepharose column chromatography. Protein sequencing and mass spectrometry demonstrated that chymotrypsin cleaved the N-terminal 6, 15, or 23 amino acids (T6, T15, and T23). The blue-shifts of the absorption maxima and the increases in the apparent pK(a) of the chromophores relative to those of intact PYP were less than 4 nm and 0.2, respectively. The absorption spectra of the near-UV intermediates produced from T6, T15, and T23 were identical to that of intact PYP, but with lifetimes that were 140, 2,300, and 4,500 times longer, respectively. These observations suggest that the recovery of the dark state of PYP from the near-UV intermediate is accelerated by the N-terminal region, and that this region acts as a regulatory factor for the photocycle of PYP.