The Light Growth Response of Phycomyces

With the help of an automated tracking system we have studied the characteristics of the transient light growth response of Phycomyces. The response shows a sharply defined latency. The Q₁₀ of the reciprocal latency is 2.4. Response patterns at different peaks of the action spectrum are the same. The gradual variation of response magnitude over a wide range of adapted intensifies parallels that of phototropism. The responses to saturating stimuli exhibit a strong oscillation with a constant period of 1.6 min and variable damping. The growth responses to sinusoidally varying light intensities show a system bandwidth of 2.5 x 10^-3 Hz. The linear dependence of phase shift on frequency is largely attributable to the latency observed with pulse stimuli. In the high intensity range a previously suspected increase of the steady-state growth rate with intensity has been confirmed. The light growth responses of mutants selected for diminished phototropism have been investigated. Many of these mutants have sizable but grossly distorted growth responses.     

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.     

Characterization of Staphylococcus epidermidis mutants sensitive to ultraviolet radiation

Five UV-sensitive mutants obtained by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) treatment of the Staphyloccocus epidermidis W5 strain were characterized phenotypically by assaying their UV- and MNNG-sensitivities, lysogenic inducibility, host-cell reactivation and Weigle reactivation capacities. The results were compared with those of well-characterized Escherichia coli strains, permitting the identification of: 2 mutants that behave as Uvr⁻ Umu⁻; 1 mutant that appears analogous to Uvr⁻; 1 mutant that resembles LexA⁻ and 1 mutant that exhibits a RecA⁻ phenotype. The study of these mutants can contribute to the understanding of the repair mechanisms in S. epidermidis.     

Phycomyces: Phototropism and light-growth response to pulse stimuli

The relationship between phototropism and the light-growth response of Phycomyces blakesleeanus (Burgeff) sporangiophores was investigated. After dark adaptation, stage-IVb sporangiophores were exposed to short pulses of unilateral light at 450 nm wavelength. The sporangiophores show a complex reaction to pulses of 30 s duration: maximal positive bending at 3·10^-4 and 10^-1 J m^-2, but negative bending at 30 J m^-2. The fluence dependence for the light-growth response also is complex, but in a different way than for phototropism; the first maximal response occurs at 1.8·10^-3 J m^-2 with a lesser maximum at 30 J m^-2. A hypertropic mutant, L85 (madH), lacks the negative phototropism at 30 J m^-2 but gives results otherwise similar to the wild type. The reciprocity rule was tested for several combinations of fluence rates and pulse durations that ranged from 1 ms to 30 s. Near the threshold fluence (3·10^-5 J m^-2), both responses increase for pulse durations below 67 ms and both have an optimum at 2 ms. At a fluence of 2.4·10^-3 J m^-2, both responses decrease for pulse durations below 67 ms. The hypertropic mutant (madH), investigated for low fluence only, gave similar results. In both strains, the time courses for phototropism and light-growth response, after single short pulses of various durations, show no clear correlation. These results imply that phototropism cannot be caused by linear superposition of localized light-growth responses; rather, they point to redistribution of growth substances as the cause of phototropism.     

Blue Light Reception in Phycomyces: Red Light Sensitization in madC Mutants

Sporangiophores of the zygomycete fungus Phycomyces blakesleeanus are sensitive to near UV and blue light. The quantum effectiveness of yellow and red light is more than 6 orders of magnitude below that of near UV or blue light. Phototropism mutants with a defect in the gene madC are about 10⁶ times less sensitive to blue light than the wild type. These mutants respond, however, to yellow and red light when the long wavelength light is given simultaneously with actinic blue light. In the presence of yellow or red light the photogravitropic threshold of madC mutants is lowered about 100-fold though the yellow and the red light alone are phototropically ineffective. A step-up of the fluence rate of broad-band red light (> 600 nm) from 6 × 10^?3 to 6W m^?2 elicits, in mutant C 148 madC, a transient deceleration of the growth rate. The growth rate of the wild type is not affected by the same treatment. The results are interpreted in terms of a red light absorbing intermediate of the blue light photoreceptor of Phycomyces. The intermediate should be short-lived in the wild type and should accumulate in madC mutants.     

System analysis of Phycomyces light-growth response: Single mutants

The white-noise method of system identification has been applied to the transient light-growth response of a set of seven mutants of Phycomyces with abnormal phototropism, affected in genes madA to madG. The Wiener kernels, which represent the input-output relation of the light-growth response, have been evaluated for each of these mutants and the wild-type strain at a log-mean blue-light intensity of 0.1 W m^-2. Additional experiments were done at 3x10^-4 and 10 W m^-2 on the madA strain C21 and wild-type. In the normal intensity range (0.1 W m^-2) the madA mutant behaves similarly to wild-type, but, at high intensity, the madA response is about twice as strong as that of wild-type. Except for C21 (madA), the first-order kernels of all mutants were smaller than the wild-type kernel. The first-order kernels for C111 (madB) and L15 (madC) show a prolonged time course, and C111 has a longer latency. The kernels for C110 (madE), C316 (madF), and C307 (madG) have a shallow and extended negative phase. For C68 (madD), the latency and time course are shorter than in the wild-type. These features are also reflected in the parameters estimated from fits of the anlytical model introduced in the previous paper to the experimental transfer functions (Fourier transforms of the kernels). The kernel for L15 (madC) is described better by a model that lacks one of the two second-order low-pass filters, because its response kinetics are dynamically of lower order.     

Carotenoid Mutants of Mucor circinelloides

The wild-type of the filamentous fungus Mucor circinelloides accumulates the yellow pigment β-carotene. At a continuous blue-light fluence rate of 0.1 W/m² the β-carotene content increases about eight fold over the dark controls. Among the mutants isolated after exposure of spores to either N-methyl-N'-nitro-N-nitrosoguanidine or ICR-170, a red mutant accumulating lycopene, white mutants accumulating phytoene and white mutants without carotenoids were found. The biosynthesis of carotenoids in M. circinelloides shows similarities with that of the fungus Phycomyces blakesleeanus such as the presence of mutants in the same structural genes and the induction by light of the pathway. However, negative end-product regulation by β-carotene on the biosynthetic pathway, as in Phycomyces, is absent in M. circinelloides. In contrast to Phycomyces carB and carR mutants, carotenoids in corresponding mutants of M. circinelloides are photoinduced.     

Pulse amplitude modulated fluorescence in the green macrophytes,Codium adherens,Enteromorpha muscoides,Ulva gigantea and Ulva rigida,from the Atlantic coast of Southern Spain

The effect of ambient and enhanced solar radiation on the photosynthetic apparatus in four marine green macroalgae on the Southern coast of Spain (Strait of Gibraltar) was investigated using pulse amplitude modulation (PAM) fluorescence. The dependence of the fluorescence parameters on the irradiance of the actinic light was determined for all four species. It showed that maximal fluorescence after light adaptation (F_m′), photochemical quenching (q_P) and the photosynthetic quantum yield decreased in Enteromorpha muscoides with irradiance while non-photochemical quenching (q_N) rose continuously. In Ulva rigida the photosynthetic quantum yield dropped at irradiances above 4 W m⁻² but q_P did not decrease with increasing light. q_N quenching rose sharply above 37 W m⁻², and maximal fluorescence dropped above 1 W m⁻². In Ulva gigantea the yield dropped to zero at irradiances of 37 W m⁻², as did q_P at 53 W m⁻². q_N started from an intermediate level and increased to a maximum at the highest irradiances. In Codium adherens, the yield and q_P behaved similarly as in U. rigida, while q_N rose at much lower irradiances. All investigated algae suffered from photoinhibition even at their natural sites of growth when the sun is at high angles. The hypothesis that algae with flat thalli suffer more than those with massive ones was confirmed. Photoinhibition was less pronounced in U. rigida and C. adherens than in the other two species. After 1 h of exposure to solar radiation at the surface, the photosynthetic quantum yield decreased substantially in the surface algae E. muscoides and U. rigida. In both macroalgae, recovery of the photosynthetic quantum yield was almost complete after 2–3 h in the shade. Two other green algae from shaded habitats (U. gigantea and C. adherens) did not show complete recovery of the yield from photoinhibition. This confirms the second hypothesis that sun-adapted algae recover faster from photoinhibition than those adapted to shaded sites.     

Domain-specific determinants of catalysis/substrate binding and the oligomerization status of barley UDP-glucose pyrophosphorylase

UDP-glucose (UDPG) pyrophosphorylase (UGPase) produces UDPG for sucrose and polysaccharide synthesis and glycosylation reactions. In this study, several barley UGPase mutants were produced, either single amino acid mutants or involving deletions of N- and C-terminal domains (Ncut and Ccut mutants, respectively) and of active site region (“NB loop”). The Del-NB mutant yielded no activity, whereas Ncut deletions and most of Ccut mutants, including short deletions at the so called “I-loop” region of C-terminal domain, as well as a single K260A mutant resulted in very low activity. For wt and the mutants, kinetics with UDPG were linear on reciprocal plots, whereas PPi at concentrations above 1 mM exerted strong substrate inhibition. Both K260A and most of the Ccut mutants had very high K_m with PPi (up to 33 mM), whereas Ncut deletions had greatly increased K_m with UDPG (up to 57 mM). Surprisingly, an 8 amino acid deletion from end of the C-terminus resulted in an enzyme (Ccut-8 mutant) with 44% higher activity when compared to wt, but with similar K_m values. Whereas Ccut-8 existed solely as a monomer, other deletion mutants had a more oligomerized status, e.g. Ncut mutants existing primarily as dimers. Overall, the data confirmed the essential role of NB loop in catalysis, but also pointed out to the role of both N- and C-termini for activity, substrate binding and oligomerization. The importance of oligomerization status for enzymatic activity of UGPase is discussed.     

Phototropism and polarotropism of primary chloronemata of the moss Physcomitrella patens: responses of mutant strains

The phototropic and polarotropic responses of primary chloronemata grown from germinated minated spores of three mutant strains of the moss, Physcomitrella patens, have been studied and compared with those of the wild-type. The mutants and wild-type show the same qualitative tropic responses but differ with respect to the light conditions under which they are expressed. In both the wild-type and mutants the responses are controlled by phytochrome. In monochromatic red light, at low fluence rates, wild-type primary chloronemata grow positively phototropically in unidirectional light or perpendicular to the electrical vector (E) in polarised light; at high fluence rates growth in unidirectional light is lateral to the incident light or, in polarised light, parallel to E. The mutants, however, show only the lateral phototropic or parallel polarotropic responses at all fluence rates of red light tested. In far-red light, the wild-type primary chloronemata adopt a positive phototropic or a perpendicular polarotropic response; the mutants show the same responses but in a lower percentage of filaments. These results and those at other wavelengths indicate either that the mutants are impaired in their ability to adopt the positive phototropic and perpendicular polarotropic responses or that in the mutants the transition between the “low light” (positive phototropic-perpendicular polarotropic) and the “high light” (lateral phototropic-parallel polarotropic) responses is shifted to a lower photon fluence rate. Possible explanations of this phenotypic difference are discussed.     

The menD and menE homologs code for 2-succinyl-6-hydroxyl-2,4-cyclohexadiene-1-carboxylate synthase and O-succinylbenzoic acid–CoA synthase in the phylloquinone biosynthetic pathway of Synechocystis sp. PCC 6803

The genome of the cyanobacterium Synechocystis sp. PCC 6803 contains genes identified as menD and menE, homologs of Escherichia coli genes that code for 2-succinyl-6-hydroxyl-2,4-cyclohexadiene-1-carboxylate (SHCHC) synthase and O-succinylbenzoic acid–CoA ligase in the menaquinone biosynthetic pathway. In cyanobacteria, the product of this pathway is 2-methyl-3-phytyl-1,4-naphthoquinone (phylloquinone), a molecule used exclusively as an electron transfer cofactor in Photosystem (PS) I. The menD⁻ and menE⁻ strains were generated, and both were found to lack phylloquinone. Hence, no alternative pathways exist in cyanobacteria to produce O-succinylbenzoyl–CoA. Q-band EPR studies of photoaccumulated quinone anion radical and optical kinetic studies of the P700⁺ [F_A/F_B]⁻ backreaction indicate that in the mutant strains, plastoquinone-9 functions as the electron transfer cofactor in the A₁ site of PS I. At a light intensity of 40 μE m⁻² s⁻¹, the menD⁻ and menE⁻ mutant strains grew photoautotrophically and photoheterotrophically, but with doubling times slower than the wild type. Both of which are sensitive to high light intensities. Low-temperature fluorescence studies show that in the menD⁻ and menE⁻ mutants, the ratio of PS I to PS II is reduced relative to the wild type. Whole-chain electron transfer rates in the menD⁻ and menE⁻ mutant cells are correspondingly higher on a chlorophyll basis. The slower growth rate and high-light sensitivity of the menD⁻ and menE⁻ mutants are therefore attributed to a lower content of PS I per cell.     

Negative phototropism in the piloboloid mutants of Phycomyces blakesleeanus Bgff.

The sporangiophore (spph) of a piloboloid mutant, genotype pil, of Phycomyces ceases elongation and expands radially in the growth zone shortly after reaching the developmental stage IV b. The pil spph is always negatively phototropic to unilateral visible light when its diameter exceeds 210 m. Photoinduction of spph initiation, light-growth response, threshold of light energy fluence rate for the negative phototropism, avoidance and gravitropism in the pil mutant are all normal. In liquid paraffin, the pil spph shows negative phototropism as does the wild-type spph. Genetic analyses indicate that the negative phototropism of the pil mutant is governed by the phenotypic characteristics of pil but not by specific gene(s) responsible for negative phototropism. These facts imply that the reverse phototropism of the pil mutant results from a loss of the convergent lens effect of the cell because of the increase in cell diameter.Abbreviations spph(s) sporangiophore(s) - wt(s) wild type(s)     

Light intensity increases the susceptibility of Vallisneria natans to snail herbivory

Palatability to snail herbivory (Radix swinhoei H. Adams) and C/N ratios were assessed for Vallisneria natans (Lour.) Hara, in three different experimental light regimes (midday fluxes respectively 280 μmol m⁻² s⁻¹, 15 μmol m⁻² s⁻¹, and a variable intensity between these two). Higher light intensity as well as prolonged photoperiods increased palatability and growth, and improved C/N ratio by decreasing N content. Snail growth was slightly increased but juvenile survivorship decreased under higher light. The results suggest that the availability of light may affects intraspecific variation in palatability of V. natans.     

Electrophoretic analysis of proteins from night-blind mutants of Phycomyces

Using two-dimensional gel electrophoresis, we have analyzed proteins from a plasma membrane-enriched fraction from Phycomyces sporangiophores. Specifically, we have compared gels for night-blind mutants and a wild-type strain to find proteins involved in the early steps of the sensory transduction chain for phototropism. In the gels for a mutant affected in the gene madA, a protein spot [51 kilodaltons (kdal) and pI 6.35] appears that is absent from the wild-type and the other mad mutants. Mutants affected in either of two madB alleles lack a protein spot (57 kdal and pI 6.6) that is present in the wild-type and all other mad strains; this spot probably represents the madB gene product. In some madC mutants, two spots (59 kdal, pI 6.5, with a covalently linked flavin; and 50 kdal, pI 6.4) are absent; however, in other madC strains, one or both of these spots are present. These four protein spots that are altered in madA, madB, and madC mutants may represent components of the photoreceptor complex responsible for phototropism in Phycomyces.This work was supported in part by an equipment grant to JAP from the Syracuse University Senate Research Committee, research grants to EDL from the National Science Foundation (PCM-8003915 and DMB-8316458), and a fellowship to EDL from the Alfred P. Sloan Foundation.     

Effect of Light on Uredospore Germination and Germ Tube Growth of Soybean Rust (Phakopsora pachyrhizi Syd.)

The effect of light on uredospore germination and germ tube growth of Phakopsora pachyrhizi was studied. Frequency of uredospore germination was only partially reduced by high light intensity (> 1,9 * 10⁴ mW * m^?2). In uredospores unilaterally irradiated with polychromatic light germ tubes always emerged from the shadowed side. Already developed germ tubes showed a negative phototropic response. Both effects were inducible by low light intensities. Negative phototropism of germ tubes was a blue light effect. Light of 441 nm was more effective than that of 422 nm or 372 nm. Red light (> 600 nm) was ineffective, green light (513 nm) induced medium responses. In half-side illumination studies longitudinal halves of germ tube tips and spores were irradiated under a microscope. The tips of the germ tubes bent into the illuminating beam. In half-side illumination studies germ tubes always emerged from the illuminated spore halves. Under unilateral illumination liquid paraffin reversed this light “polarization” of spores and the negative phototropism of germ tubes. These results suggest that during unilateral illumination spores and germ tube tips act as a lens focussing the light on the wall farthest away from the light source., There, in uredospores emergence of germ tubes is stimulated and in germ tubes growth is inhibited. As a consequence, under unilateral illumination germ tubes emerge at the shadowed side of the spores and grow away from the light.     

Mutants of Serratia marcescens lacking cyclic nucleotide phosphodiesterase activity and requiring cyclic 3′,5′-AMP for the utilization of various carbohydrates

Adenine requiring mutants of Serratia marcescens SM-6-F'lac ⁺ have been found to grow well in minimal-glucose medium solely supplemented with cAMP. From one of these ade strains double mutants (called ade cpd) were isolated which could no longer utilize cAMP but which still grew on 5′AMP. Dialyzed cell extracts (soluble fraction) of the double mutants, assayed for cAMP phosphodiesterase, were unable to hydrolyze cAMP whereas cell extracts of the parental strains yielded 5′AMP at a rate of 1.6–2.0 μmoles min⁻¹ mg⁻¹ protein. The loss of the phosphodiesterase activity in S. marcescens cpd W1181 did not cause an accumulation of large amounts of cAMP as was found for the diesterase-negative mutant AB257^pc-1 of Escherichia coli. The induced synthesis of β-galactosidase in mutant cpd W 1181 showed about the same sensitivity to transient and permanent catabolite (glucose) repression as the corresponding cpd ⁺ strain. Starting from S. marcescens cpd W1181 three independent double mutants (called cpd cya) were isolated which required exogenous cAMP for utilizing various carbohydrates as carbon source, for motility and for the formation of extracellular lipase and the red pigment prodigiosine. The intracellular concentration of cAMP in these mutants, grown in nutrient broth, was 40–60% of that of the parental strain which is about 4×10⁻⁴ M. However, the adenylate cyclase in cell extracts of the mutants W1237 and W1270 was like that of the corresponding cya ⁺ strain (about 2×10⁻² μmoles min⁻¹ mg⁻¹ protein).     

Characterization of Tn5-induced symbiotically defective mutants of cowpea rhizobia

Symbiotically defective mutants of cowpea rhizobia strain IRC256 were isolated by random Tn5 mutagenesis and characterized. One auxotroph (MS1) requiring adenine and thiamine was a non-nodulating mutant (Nod⁻) and three prototrophic mutants were Nod⁺ Fix⁻ which formed small and ineffective nodules on cowpeas (Vigna unguiculata). Acetylene reduction activity of the Nod⁺ Fix⁻ mutants was reduced to 80–94% of that of the wild-type strain. The non-nodulating mutant (MS1) induced root-hair curling but did not show any nodule initiation or nodule development. Ultrastructural examination of nodules formed by Fix⁻ mutants showed that these contained few bacteroids, indicating either early senescence or a reduction in bacterial release into the cytoplasm of the host cell. DNA hybridization of total DNAs from a representative number of Tn5 mutants showed that each of them had one copy of the transposon Tn5 which was randomly inserted into the genome of cowpea rhizobia.     

Mechanisms of bovine liver glutamate dehydrogenase self-association. I. Kinetic evidence for a random association of polymer chains.

We have undertaken a study of the mechanism of bovine liver glutamate dehydrogenase self-association with scattered light temperature-jump and stopped-flow relaxation techniques. Our results indicate a “random association” mechanism in which association-dissociation reactions occur between all polymerized forms of the oligomer according to

where the specific rate-constants k_a and k_d are independent of chain length. At 15 °C we find k_a = 1.5 × 10⁶m⁻¹s⁻¹ and k_d = 5 s⁻¹. Standard thermodynamic functions and activation parameters have been determined from equilibrium and kinetic experiments at different temperatures. Large entropy effects and heat capacities indicate water participation in the self-aggregation process. We suggest that the rate-determining step in the association of glutamate dehydrogenase molecules is the “melting” of a layer of ordered water structure between two hydrophobic contact sites.     

Yeast mutants sensitive to trimethoprim

Wild-type strains of Saccharomyces cerevisiae are resistant to growth inhibition by the folate antagonist trimethoprim. A mutant strain sensitive to trimethoprim was isolated. It was found to be sensitive to both ultraviolet light and X-irradiation. Genetic tests revealed that it was allelic with a known radiation-sensitive strain of Saccharomyces cerevisiae, rad 6-1. Strains harbouring a variety of mutant alleles conferring radiation-sensitivity were tested for sensitivity to trimetroprim. It was found that rad 6-1 and each of the four known alleles of rad 18 conferred sensitivity to the drug, but all other rad mutants tested were trimethoprim-resistant. All trimethoprim-sensitive strains, including double mutants of rad 6 rad 18, gave rise to trimethoprim-resistant outgrowths at a rather high frequency (∼ 10⁻⁵). Several resistant outgrowths were analysed. A wide variation in phenotype with respect to UV-sensitivity was found. Genetical analysis revealed that resistance to trimethoprim resulted from torward mutations at separate loci rather than back mutations of rad 6 or rad 18 alleles.