Growth rate fluctuations in phycomyces sporangiophores

The growth rate of the Phycomyces sporangiophore fluctuates under constant environmental conditions. These fluctuations underlie the well-characterized sensory responses to environmental changes. We compared growth fluctuations in sporangiophores of unstimulated wild type and behavioral mutants by use of maximum entropy spectral analysis, a mathematical technique that estimates the frequency and amplitude of oscillations in a time series. The mutants studied are believed to be altered near the input (“night-blind”) or output (“stiff” and “hypertropic”) of the photosensory transduction chain. The maximum entropy spectrum of wild type shows a sharp drop-off in spectral density above 0.3 millihertz, several minor peaks between 0.3 and 10 millihertz, and a broad maximum near 10 millihertz. Similar spectra were obtained for a night-blind mutant and a hypertropic mutant. In contrast, the spectra of three stiff mutants, defective in genes madD, madE, or madG, had distinctive peaks near 1.6 mHz and harmonics of this frequency. A madF stiff mutant, which is less stiff than madD, madE, and madG mutants, had a spectrum intermediate between wild type and the three other stiff mutants. Our results indicate that alterations in one or more steps associated with growth regulation output cause the Phycomyces sporangiophore to express a rhythmic growth rate.     

Growth of the morphological,piloboloid mutants ofPhycomyces blakesleeanus

The sporangiophore of a growth zone-defective mutant, piloboloid (genotypepil), ofPhycomyces gradually ceases longitudinal elongation and radially expands until the growth zone becomes nearly spherical, shortly after the sporangiophore has reached the final developmental stage. The growth of 23pil mutants, obtained by mutagenesis withN-methyl-N′-nitro-N-nitrosoguanidine (NTG), 2-methoxy-6-chloro-9-[3-(ethyl-2-chloroethyl)aminopropylamino]acridine-2HCl (ICR-170), or 4-nitroquinoline-1-oxide (NQO), was studied under different conditions. Allpil mutants obtained were genetically homokaryotic. Expansion in the growth zone and degree of synchrony ofpil sporangiophore length were both greater at low temperature (10°C) and under white light (10 μW/cm²) than at high temperature (20, 25°C) and in the dark. Dwarf sporangiophores of about 1-mm length, which characteristically stop growing immediately after sporangium formation, did not expand radially. The mycelial morphology ofpil mutants was almost normal compared with that of the wild type, though growth was some-what slower in the former than the latter.     

Linear Dichroism and Orientation of the Phycomyces Photopigment

The greater sensitivity of a cylindrical Phycomyces sporangiophore to blue light polarized transversely rather than longitudinally is a consequence of the dichroism and orientation of the receptor pigment. The abilities of wild type and several carotene mutants to distinguish between the two directions of polarization are the same. The E-vector angle for maximum response relative to the transverse direction is 42 ± 4° at 280 nm, 7° ± 3° at 456 nm, and 7° ± 8° at 486 nm. The in vivo attenuation of polarized light at these wavelengths is very small. The polarized light effect in Phycomyces cannot arise from reflections at the cell surface or from differential attenuations due to internal screening or scattering.     

Mutants ofPhycomyces with enhanced tropisms

Seven mutants ofPhycomyces which exhibit phototropism at high intensity of blue light (10 W/m²) where the wild-type strain is unresponsive have been isolated. These mutants have the same absolute threshold for phototropism as wild type (10⁻⁹ W/m²). In comparison to wild type in the region just above this threshold, the mutants respond more strongly to light than to gravity, as determined by photogeotropic equilibrium experiments. The kinetics of phototropism, avoidance, and geotropism are also enhanced in the mutants. Therefore these mutants are designated phenotypically by the term “hypertropic.” The phenotype of these mutants is in many ways opposite to that of the so-called “stiff” mutants, which have slow tropisms. The rapid bending rates of the hypertropic mutants may be associated in part with the smaller diameter of their sporangiophores, but not according to a simple proportional relationship between phototropic bending rate and inverse diameter that applies for wild type and the hypergeotropic mutant C5. For photophorogenesis, which is mediated by the mycelium, one hypertropic mutant studied responds similarly to wild type. The pleiotropic character of these mutants suggests that they are affected near the output of the common sensory transduction pathway for these responses.     

Light and dark adaptation in Phycomyces phototropism

Light and dark adaptation of the phototropism of Phycomyces sporangiophores were analyzed in the intensity range of 10(-7)-6 W X m- 2. The experiments were designed to test the validity of the Delbruck- Reichardt model of adaptation (Delbruck, M., and W. Reichardt, 1956, Cellular Mechanisms in Differentiation and Growth, 3-44), and the kinetics were measured by the phototropic delay method. We found that their model describes adequately only changes of the adaptation level after small, relatively short intensity changes. For dark adaptation, we found a biphasic decay with two time constants of b1 = 1-2 min and b2 = 6.5-10 min. The model fails for light adaptation, in which the level of adaptation can overshoot the actual intensity level before it relaxes to the new intensity. The light adaptation kinetics depend critically on the height of the applied pulse as well as the intensity range. Both these features are incompatible with the Delbruck-Reichardt model and indicate that light and dark adaptation are regulated by different mechanisms. The comparison of the dark adaptation kinetics with the time course of the dark growth response shows that Phycomyces has two adaptation mechanisms: an input adaptation, which operates for the range adjustment, and an output adaptation, which directly modulates the growth response. The analysis of four different types of behavioral mutants permitted a partial genetic dissection of the adaptation mechanism. The hypertropic strain L82 and mutants with defects in the madA gene have qualitatively the same adaptation behavior as the wild type; however, the adaptation constants are altered in these strains. Mutation of the madB gene leads to loss of the fast component of the dark adaptation kinetics and to overshooting of the light adaptation under conditions where the wild type does not overshoot. Another mutant with a defect in the madC gene shows abnormal behavior after steps up in light intensity. Since the madB and madC mutants have been associated with the receptor pigment, we infer that at least part of the adaptation process is mediated by the receptor pigment.     

Isolation and Characterization of Two Cellulose Morphology Mutants of Gluconacetobacter hansenii ATCC23769 Producing Cellulose with Lower Crystallinity

Gluconacetobacter hansenii, a Gram-negative bacterium, produces and secrets highly crystalline cellulose into growth medium, and has long been used as a model system for studying cellulose synthesis in higher plants. Cellulose synthesis involves the formation of β-1,4 glucan chains via the polymerization of glucose units by a multi-enzyme cellulose synthase complex (CSC). These glucan chains assemble into ordered structures including crystalline microfibrils. AcsA is the catalytic subunit of the cellulose synthase enzymes in the CSC, and AcsC is required for the secretion of cellulose. However, little is known about other proteins required for the assembly of crystalline cellulose. To address this question, we visually examined cellulose pellicles formed in growth media of 763 individual colonies of G. hansenii generated via Tn5 transposon insertion mutagenesis, and identified 85 that produced cellulose with altered morphologies. X-ray diffraction analysis of these 85 mutants identified two that produced cellulose with significantly lower crystallinity than wild type. The gene disrupted in one of these two mutants encoded a lysine decarboxylase and that in the other encoded an alanine racemase. Solid-state NMR analysis revealed that cellulose produced by these two mutants contained increased amounts of non-crystalline cellulose and monosaccharides associated with non-cellulosic polysaccharides as compared to the wild type. Monosaccharide analysis detected higher percentages of galactose and mannose in cellulose produced by both mutants. Field emission scanning electron microscopy showed that cellulose produced by the mutants was unevenly distributed, with some regions appearing to contain deposition of non-cellulosic polysaccharides; however, the width of the ribbon was comparable to that of normal cellulose. As both lysine decarboxylase and alanine racemase are required for the integrity of peptidoglycan, we propose a model for the role of peptidoglycan in the assembly of crystalline cellulose.     

Phycomyces: distribution of growth velocities in the growing zone

Distribution of growth velocities in the growing zone of stage IVb Phycomyces sporangiophores was measured by photographing the growing zone after dusting it with starch grains. When the entire growing zone is fully dark-adapted to red light and then subjected to a saturating white light stimulus, the entire growing zone increases in growth rate. When the growing zone is partially light-adapted, again the entire growing zone responds when subjected to a saturating white light stimulus but to a lesser degree than the fully dark-adapted sporangiophore. Phototropic mutants of class 1 and class 2 show a distribution of growth in the growing zone similar to wild type sporangiophores both during steady-state growth and during light-stimulated growth.     

Specific tropism caused by ultraviolet C radiation in Phycomyces

The giant sporangiophores of Phycomyces blakesleeanus turn towards blue and away from ultraviolet C sources (wavelength under 310 nm). We have isolated fifteen mutants with normal blue tropism but defective ultraviolet tropism. Wild-type sporangiophores described a double turn when exposed successively to blue and ultraviolet beams coming from the same side; under certain conditions, the mutants turned only to the blue. The new uvi mutations modified the behaviour in heterokaryosis and were lethal in homokaryosis, i.e., they affected essential cellular components. The responses of the wild type and one of the mutants were registered and evaluated with a computer-aided device. The mutant behaved normally under blue light, but took longer than the wild type to turn away from the ultraviolet source. With very weak ultraviolet stimuli (10(-8) and l0(-9) W m-2), the wild type turned towards the source, but the mutant did not respond. Calculations of absorbed-energy distributions in the sporangiophore showed that Phycomyces responds differently to similar spatial distributions of blue and ultraviolet radiations. Wild-type and mutant sporangiophores had the same high ultraviolet absorption due to gallic acid. We conclude that ultraviolet tropism is not just a modification of blue phototropism due to the high ultraviolet absorption of the sporangiophores. Phycomyces has a separate sensory system responsive to ultraviolet radiation, but not to blue light.     

OCTAHEDRAL CRYSTALS IN PHYCOMYCES. II

"Phycomyces blakesleeanus" sporangiophores contain octahedral crystals throughout their cytoplasm and vacuole. More octahedral crystals were found in the wild-type strain G5 (+) than in the β-carotene-deficient mutant C5 (-), and much more than in the mutant C141 (-), which is sensitive to only high light intensity. In the wild type, the number of crystals per sporangiophore increased until the sporangiophore reached stage IV, and then decreased. Stage I contained the most crystals per unit volume. Cultures grown in darkness had the maximum number of crystals. Under high light intensity, there was an overall reduction of crystals. The crystals are regular octahedrons. The crystals were isolated from the sporangiophores by a method of sucrose density-gradient centrifugation. They contain nearly 95% protein, are stable in organic solvents, but can be solubilized in buffer solution above pH 9.5 and below 2.5. The crystals weakly fluoresce with an emission peak at 540 nm, which is affected by irradiation with white light. Absorption spectra of freshly prepared crystals show absorption maxima around 265–285 nm, 350–380 nm, and 450–470 nm. These absorption peaks for the crystals are close to those of the phototropic and light-growth action spectra. These data suggest that the crystals may contain a flavoprotein which may be the photoreceptor pigment of "Phycomyces".     

Roles of Chemosensory Pathways in Transient Changes in Swimming Speed of Rhodobacter sphaeroides Induced by Changes in Photosynthetic Electron Transport

The response of free-swimming Rhodobacter sphaeroides to increases and decreases in the intensity of light of different wavelengths was analyzed. There was a transient (1 to 2 s) increase in swimming speed in response to an increase in light intensity, and there was a similar transient stop when the light intensity decreased. Measurement of changes in membrane potential and the use of electron transport inhibitors showed that the transient increase in swimming speed, following an increase in light intensity, and the stop following its decrease were the result of changes in photosynthetic electron transport. R. sphaeroides has two operons coding for multiple homologs of the enteric chemosensory genes. Mutants in the first chemosensory operon showed wild-type photoresponses. Mutants with the cheA gene of the second operon (cheA_II) deleted, either with or without the first operon present, showed inverted photoresponses, with free-swimming cells stopping on an increase in light intensity and increasing swimming speed on a decrease. These mutants also lacked adaptation. Transposon mutants with mutations in cheA_II, which also reduced expression of downstream genes, however, showed no photoresponses. These results show that (i) free-swimming cells respond to both an increase and a decrease in light intensity (tethered cells only show the stopping on a step down in light intensity), (ii) the signal comes from photosynthetic electron transfer, and (iii) the signal is primarily channelled through the second chemosensory pathway. The different responses shown by the cheA_II deletion and insertion mutants suggest that CheW_II is required for photoresponses, and a third sensory pathway can substitute for CheA_II as long as CheW_II is present. The inverted response suggests that transducers are involved in photoresponses as well as chemotactic responses.     

Light response inPhycomyces blakesleeanus: Evidence for roles of chitin biosynthesis and breakdown

Chitin synthetase activity, both basal and zymogenic, fromPhycomyces sporangiophores was stimulated by lightin vitro andin vivo. AmadB mutant did not display these activations, whereas in amadE mutant only chitin synthetase zymogen was increased by illuminationin vivo. Light also produced a transient alteration in cell wall structure at the apical region of the sporangiophore revealed by accessibility of chitin to binding by wheat germ agglutinin and by an increased limited breakage of chitin microfibrils. This last response was absent in bothmadB andmadE mutants. Accordingly, it is suggested that the light growth response in the sporangiophore fromPhycomyces is due to a transient softening of the cell wall at the growing region followed by an elongation due to the turgor pressure of the cell and an enhanced chitin biosynthesis by the apically localized chitin synthetase which restores normal strength to the cell wall. A hypothetical scheme to account for these results is presented.     

Loss of CpSRP54 function leads to a truncated light-harvesting antenna size in Chlamydomonas reinhardtii

The Chlamydomonas reinhardtii truncated light-harvesting antenna 4 (tla4) DNA transposon mutant has a pale green phenotype, a lower chlorophyll (Chl) per cell and a higher Chl a/b ratio in comparison with the wild type. It required a higher light intensity for the saturation of photosynthesis and displayed a greater per chlorophyll light-saturated rate of oxygen evolution than the wild type. The Chl antenna size of the photosystems in the tla4 mutant was only about 65% of that measured in the wild type. Molecular genetic analysis revealed that a single plasmid DNA insertion disrupted two genes on chromosome 11 of the mutant. A complementation study identified the “chloroplast signal recognition particle 54” gene (CpSRP54), as the lesion causing the tla4 phenotype. Disruption of this gene resulted in partial failure to assemble and, therefore, lower levels of light-harvesting Chl-binding proteins in the C. reinhardtii thylakoids. A comparative in silico 3-D structure-modeling analysis revealed that the M-domain of the CpSRP54 of C. reinhardtii possesses a more extended finger loop structure, due to different amino acid composition, as compared to that of the Arabidopsis CpSRP54. The work demonstrated that CpSRP54 deletion in microalgae can serve to generate tla mutants with a markedly smaller photosystem Chl antenna size, improved solar energy conversion efficiency, and photosynthetic productivity in high-density cultures under bright sunlight conditions.     

An Increase in Mechanical Extensibility during the Period of Light-stimulated Growth

The sporangiophore of Phycomyces responds to a temporary increase in light intensity with a transient increase in growth rate that begins 2 to 3 minutes after the initiation of the stimulus and continues until approximately the 12th minute. Tensile tests conducted on the stage IVb sporangiophore demonstrate that an increase in mechanical extensibility of the cell wall occurs 2 minutes after the initiation of a light stimulus and continues until approximately the 15th minute. This finding supports the theory that light-stimulated plant cell expansion and rate of expansion is a function of the mechanical extensibility of the cell wall.     

Modified Light-Induced Absorbance Changes in dim Y Photoresponse Mutants of Trichoderma

A brief pulse of blue light induces the common soil fungus Trichoderma harzianum to sporulate. Photoresponse mutants with higher light requirements than the wild type are available, including one class, dim Y, with modified absorption spectra. We found blue-light-induced absorbance changes in the blue region of the spectrum, in wild-type and dim Y mutant strains. The light-minus-dark difference spectra of the wild type and of several other strains indicate photoreduction of flavins and cytochromes, as reported for other fungi and plants. The difference spectra in strains with normal photoinduced sporulation have a prominent peak at 440 nm. After actinic irradiation, this 440 nanometer difference peak decays rapidly in the dark. In two dim Y photoresponse mutants, the difference spectra were modified; in one of these, LS44, the 440 nanometer peak was undetectable in difference spectra. Detailed study of the dark-decay kinetics in LS44 and the corresponding control indicated that the 440 nanometer difference peak escaped detection in LS44 because it decays faster than in the control. The action spectrum of the 440 nm difference peak is quite different from that of photoinduced sporulation. The light-induced absorbance changes are thus unlikely to be identical to the primary photochemical reaction triggering sporulation. Nevertheless, these results constitute genetic evidence that physiologically relevant pigments participate in these light-induced absorbance changes in Trichoderma.     

Arsenic tolerance in a Chlamydomonas photosynthetic mutant is due to reduced arsenic uptake even in light conditions

Arsenate resistance has been used for screening for photosynthetic mutants of Chlamydomonas, since photosynthetic mutants, such as CC981 defective in phosphoribulokinase, were shown to have arsenate resistance. Also, another type of arsenate-resistant mutants, including AR3 that lacks a homolog of a phosphate (Pi) transporter, PTB1, has been isolated. We investigated the uptake of Pi and arsenate, and the gene expression of Pi transporters, which are involved in both Pi and arsenate transport, in mutants CC981 and AR3. In the wild type, both Pi and arsenate uptake were initially high, but were inactivated in the presence of arsenate with time, especially in the dark. In contrast, both mutants were shown to exhibit higher Pi uptake, but lower arsenate uptake than the wild type, regardless of the presence or absence of light. Then, the gene expression of Pi transporters in the cells used for the uptake measurements was investigated and compared between the mutants and the wild type. In CC981, the mRNA levels of PTA2 and PTA4 were higher, while those of PTB3 and PTB5 were lower, as compared with in the wild type. In AR3, those of PTA2 and PTB2 were higher, but that of PTB5 was lower than in the wild type. These findings suggest that the arsenate resistance shown by the mutants in light is due to reduction of arsenate uptake probably through the down-regulation of some Pi transporter expression, while the Pi uptake maintained even in the dark is possibly related to higher expression of other Pi transporter(s) than in the wild type.     

Mutations perturbing petal cell shape and anthocyanin synthesis influence bumblebee perception of Antirrhinum majus flower colour

We wished to understand the effects on pollinator behaviour of single mutations in plant genes controlling flower appearance. To this end, we analysed snapdragon flowers (Antirrhinum majus), including the mixta and nivea mutants, in controlled laboratory conditions using psychophysical tests with bumblebees. The MIXTA locus controls petal epidermal cell shape, and thus the path that incident light takes within the pigment-containing cells. The effect is that mixta mutant flowers are pink in comparison to the wild type purple flowers, and mutants lack the sparkling sheen of wild type flowers that is clearly visible to human observers. Despite their fundamentally different appearance to humans, and even though bees could discriminate the flowers, inexperienced bees exhibited no preference for either type, and the flowers did not differ in their detectability in a Y-maze—either when the flowers appeared in front of a homogeneous or a dappled background. Equally counterintuitive effects were found for the non-pigmented, UV reflecting nivea mutant: even though the overall reflectance intensity and UV signal of nivea flowers is several times that of wild type flowers, their detectability was significantly reduced relative to wild type flowers. In addition, naïve foragers preferred wild type flowers over nivea mutants, even though these generated a stronger signal in all receptor types. Our results show that single mutations affecting flower signal can have profound effects on pollinator behaviour—but not in ways predictable by crude assessments via human perception, nor simple quantification of UV signals. However, current models of bee visual perception predict the observed effects very well.     

Complementation betweenPhycomyces mutants of mating type (+) with abnormal phototropism

Summary Twelve mutants ofPhycomyces blakesleeanus with defects in sporangiophore phototropism (genotypemad) were obtained from a wild type of the (+) mating type by mutagenesis with nitrosoguanidine. These mutants were tested for genetic complementation against standard (+)mad mutants derived from sexual crosses between the isogenic (+) strain and established (-)mad mutants (Ootaki et al., 1974; Eslava et al., 1976). Heterokaryons for complementation tests were obtained by grafting stage I sporangiophores. The (+) mutants were also investigated for their sensory responses such as photoinduction of sporangiophores and avoidance. The mutants were grouped into two classes, based on the phenotypic classification scheme of Bergman et al. (1973). There were eleven class 1.2 mutants and one class 2 mutant. Complementation tests revealed that all eleven class 1.2 mutants carry the genemadC and the class 2 mutant carriesmadD. There was no evidence that any were double mutants. These results are consistent with the phenotypic classification and with the complementation results of themad mutants of the (-) mating type.     

The physiological role of the carotenoid pigments of halobacterium salinarium

Summary A colorless mutant of the extremely halophilic bacterium, Halobacterium salinarium, has been isolated. In comparative experiments with the carotenoid-containing wild type, it was shown that neither type was killed upon exposure to bright sunlight under aerobic conditions. Exposure to tungsten light of an incident intensity comparable to that of bright sunlight inhibited strongly the growth of the colorless mutant; the growth of the carotenoid-containing wild type was unaffected.In their natural habitats, the extremely halophilic bacteria are usually exposed to bright sunlight. It seems likely that such light will affect the growth of these bacteria as does tungsten light. It is therefore inferred that in their natural habitats, carotenoid-containing forms will develop more freely than colorless forms, because the pigments protect the cells against an inhibitory effect of light upon growth. Carotenoid-containing forms have therefore come to dominate the population of extremely halophilic bacteria in nature.     

Oxygen Sensitive Nitrogen-Fixing Mutants of Anabaena

Two Anabaena mutants having heterocysts but incapable of fixing molecular nitrogen in air have been isolated by using ultraviolet radiation or NTG mutagenesis. Their vegetative cells differentiated into heterocysts at a higher frequency than that of the wild type. The phenotype of the mutants is stable and a low frequence of spontaneous reversion was observed. Under microaerobic condition the mutants cells can express the genetic information which encodes nitrogenase synthesis and were capable of utilizing nitrogen for growth with a low acetylene reductiop activity. The level of nitrogenase activity was correlated reciprocally with the content of cell phycocyanin and the light intensity. Both synthesis and activity of the mutant nitrogenase were very sensitive than wild type to the oxygen in vive. Introduction of 1% O2 (v/v) into the gas phase inhibited evidently acetylene reduction. Exposure of the mutant suspension to 20% O2 (v/v) resulted in total and irreversible denaturation of nitrogenase. Withdrawing of O2 in gas phase, the nitrogenase was synthesized de nero; The synthesis process was repressed by chloramphenical or ammonia. The nitrogenase activity of mutant cells increased significantly either by nitrogen- starvating to decrease the phycocyanin content or by lowering the light intensity. Specifically, during the anaerobic induction by treating the mutants filaments with diehloromethylurea which prevents photosynthetic oxygen production, the specific activity of mutant nitrogcnase was equivalent nearly to that of wild type. The ability to reduce 2, 3, 5-triphenyltetrazolium was lower in heterocysts and vegetative cells of mutants than in that of wild type. The results suggest that the oxygen sensitivity of nitrogen fixation by heterocystous bluegreen algal mutants may be duc to the defect of some enzymic systems which might play a role in scavenging oxygen toxity, so that the process of nitrogen fixation is inhibited by the active oxygen produced by vegetative cells. The mechanism of protecting nitrogenase from oxygen damage in blue-green algae is discussed.     

Control of Free Methionine Production in Wild Type and Ethionine-resistant Mutants of Chlorella sorokiniana

Mutants of Chlorella sorokiniana selected for resistance to the methionine analogue ethionine took up ethionine at the same rate as did the wild type strain. Cells of two ethionine-resistant mutants produced severalfold higher levels of free methionine and cysteine than did wild type cells.