Isolation of L-ethionine resistant cell lines in Vigna sinensis L. after mutagen treatment

Chemical mutagenesis was employed for the isolation of variant cell lines resistant to L-Ethionine (L-Eth) in Vigna sinensis L. We have measured cell survival after treatment of Vigna sinensis cell suspensions with different mutagens: Ethyl methanesulfonate (EMS), N-methyl-N-nitrosoguanidine (NTG) and Acridine Orange (AO). NTG was more toxic than EMS and AO.L-Eth resistant colonies were isolated by plating on selective medium after NTG treatment. The frequencies of appearance of resistant cells of MS-3 media supplemented with 20 g/ml L-Eth were 1.3 × 10^-6 to 1.8 × 10^-5. The highest number of L-Eth resistant calli were recorded in cells treated with 10 g/ml NTG. Few resistant colonies also appeared spontaneously from non-mutagenized cultures with a frequency of 2.9 × 10^-7 to 5.7 × 10^-7. However, a number of isolated colonies were discarded after successive retesting. The number of resistant calli dropped from 204 to 22 during successive retests. The significance of these observations has also been discussed.     

Selection and evaluation of astaxanthin-overproducing mutants of Phaffia rhodozyma

Mutagenesis of Phaffia rhodozyma with NTG yielded a mutant with an astaxanthin content of 1688 g (g dry biomass)^-1, a cell yield coefficient of 0.47 on glucose and a maximum specific growth rate of 0.12 h^-1. Re-mutation of the mutant decreased the cell yield and maximum specific growth rate but increased the astaxanthin content. The use of mannitol or succinate as carbon sources enhanced pigmentation, yielding astaxanthin contents of 1973 g g^-1 and 1926 g g^-1, respectively. The use of valine as sole nitrogen source also increased astaxanthin production, but severely decreased the maximum specific growth rate and cell yield coefficient. The optimum pH for growth of P. rhodozyma was between pH 4.5 and 5.5, whereas the astaxanthin content remained constant above pH 3.     

Genetic analysis of the phosphinothricin-tripeptide biosynthetic pathway of Streptomyces viridochromogenes Tü494

Summary Streptomyces viridochromogenes Tü494 produces the antibiotic phosphinothricyl-alanyl-alanine (Ptt). Ptt-non-producing mutants were isolated following N-methyl-N-nitro-N-nitrosoguanidine (NTG) or UV light treatment of spore suspensions. In co-synthesis and bioconversion experiments the mutational blocks in the biosynthetic pathway were localized. The mutant NTG1 was analysed in detail. This mutant acts as a secretor for all other mutants. From bioconversion experiments with presumptive precursors circumstantial evidence was obtained that NTG1 is mutated in a gene involved in the alanylation of N-acetyl-demethyl-phosphinothricin. Using a cosmid gene library the DNA region complementing the defective gene of mutant NTG1 was isolated on a 4-kb BamHI fragment. Subcloning experiments showed that a 3-kb BglII/BamHI fragment is sufficient for complementation of mutant NTG1.Formerly Susanne MüllerOffprint requests to: W. Wohlleben     

The levels of repair of endonuclease III-sensitive sites, 6-4 photoproducts and cyclobutane pyrimidine dimers differ in a point mutant forRAD14, theSaccharomyces cerevisiae homologue of the human gene defective inXPA patients

In the accompanying paper we demonstrated that endonuclease III-sensitive sites in theMAT andHML loci ofSaccharomyces cerevisiae are repaired by the Nucleotide Excision Repair (NER) pathway. In the current report we investigated the repair of endonuclease III sites, 6-4 photoproducts and cyclobutane pyrimidine dimers (CPDs) in arad14-2 point mutant and in arad14 deletion mutant. TheRAD14 gene is the yeast homologue of the human gene that complements the defect in cells from xeroderma pigmentosum (XP) patients belonging to complementation group A. In the point mutant we observed normal repair of endonuclease III sites (i.e. as wild type), but no removal of CPDs at theMAT andHML loci. Similar experiments were undertaken using the recently createdrad14 deletion mutant. Here, neither endonuclease III sites nor CPDs were repaired inMAT a orHMR a. Thus the point mutant appears to produce a gene product that permits the repair of endonuclease III sites, but prevents the repair of CPDs. Previously it was found that, in the genome overall, repair of 6-4 photoproducts was less impaired than repair of CPDs in the point mutant. The deletion mutant repairs neither CPDs nor 6-4 photoproducts in the genome overall. This finding is consistent with the RAD14 protein being involved in lesion recognition in yeast. A logical interpretation is that therad14-2 point mutant produces a modified protein that enables the cell to repair endonuclease III sites and 6-4 photoproducts much more efficiently than CPDs. This modified protein may aid studies designed to elucidate the role of the RAD14 protein in lesion recognition.     

Changes in photosynthesis and pigmentation in an agp deletion mutant of the cyanobacterium Synechocystis sp

The agp gene encoding ADP-glucose pyrophosphorylase is involved in cyanobacterial glycogen synthesis. By in vitro DNA recombination technology, agp deletion mutant (agp ^–) of cyanobacterium Synechocystis sp. PCC 6803 was constructed. This mutation led to a complete absence of glycogen biosynthesis. As compared with WT (wild type), a 60% decrease in ratio of the c-phycocyanine/chlorophyll a and no significant change in the carotenoid/chlorophyll a were observed in agp ^– cells. The agp ^– mutant had 38% less photosynthetic capacity when grown in light over 600 mol m^–2 s^–1. Under lower light intensity, the final biomass of the mutant strain was only 1.1 times of that of the WT strain under mixotrophic condition after 6 d culture. Under higher light intensity, however, the final biomass of the WT strain under mixotrophic conditions was 3 times that of the mutant strain after 6 d culture and 1.5 times under photoautotrophic conditions. The results indicate that there is a minimum requirement for glycogen synthesis for normal growth and development in cyanobacteria.     

Rearrangement of light harvesting bacteriochlorophyll homologues as a response of green sulfur bacteria to low light intensities

The pigment composition of two species of green-colored BChl c-containing green sulfur bacteria (Chlorobium limicola and C. chlorovibrioides) and two species of brown-colored BChl e-containing ones (C. phaeobacteroides and C. phaeovibrioides) incubated at different light intensities have been studied. All species responded to the reduction of light intensity from 50 to 1 Einstein(E) m^–2 s^–1 by an increase in the specific content of light harvesting pigments, bacteriochlorophylls and carotenoids. At critical light intensities (0.5 to 0.1 E m^–2 s^–1) only brown-colored chlorobia were able to grow, though at low specific rates (0.002 days^–1 mg prot^–1). High variations in the relative content of farnesyl-bacteriochlorophyll homologues were found, in particular BChl e ₁ and BChl e ₄, which were tentatively identified as [M, E] and [I, E] BChl_F e, respectively. The former was almost completely lost upon reduction of light intensity from 50 to 0.1 E m^–2 s^–1, whereas the latter increased from 7.2 to 38.4% and from 13.6 to 42.0% in C. phaeobacteroides and C. phaeovibrioides, respectively. This increase in the content of highly alkylated pigment molecules inside the chlorosomes of brown species is interpreted as a physiological mechanism to improve the efficiency of energy transfer towards the reaction center. This study provides some clues for understanding the physiological basis of the adaptation of brown species to extremely low light intensities.Abbreviations BChl bacteriochlorophyll - [M, E] BChl_F e 8-methyl, 12-ethyl BChl e, esterified with farnesol (F). Analogously: I - isobutyl - Pr propyl - Car carotenoids - Chlb chlorobactene - HPLC high performance liquid chromatography - Isr isorenieratene - LHP light harvesting pigments - PDA photodiode array detector - RC reaction center - RCH relative content of homologues     

Selection of antibiotic-resistant mutants with enhanced isoamylase activity in Pseudomonas amyloderamosa

Summary Isoamylase-hyperproducing strains of Pseudomonas amyloderamosa were bred by mutagenesis with UV light and N-methyl-N-nitro-N-nitrosoguanidine (NTG). The selection criterion for such strains was based on the formation of large turbid zones around the bacterial colonies in agar medium containing antibiotics and 1% waxy corn starch. Mutant WN6410 was obtained by treating P. amyloderamosa JD210 with five cycles of 1 × 10⁴ J UV light and one cycle of NTG. P. amyloderamosa WN6410 had 22-fold increase in isoamylase activity when compared to wild-type strain SB15 and the maximal enzyme activity, 5,100 U/ml, could be achieved within 48 h in 2.5 L fed-batch fermentation.     

Characterization of a Synechococcus sp. strain PCC 7002 mutant lacking Photosystem I. Protein assembly and energy distribution in the absence of the Photosystem I reaction center core complex

A Synechococcus sp. strain PCC 7002 psaAB::cat mutant has been constructed by deletional interposon mutagenesis of the psaA and psaB genes through selection and segregation under low-light conditions. This strain can grow photoheterotrophically with glycerol as carbon source with a doubling time of 25 h at low light intensity (10 E m^–2 s^–1). No Photosystem I (PS I)-associated chlorophyll fluorescence emission peak was detected in the psaAB::cat mutant. The chlorophyll content of the psaAB::cat mutant was approximately 20% that of the wild-type strain on a per cell basis. In the absence of the PsaA and PsaB proteins, several other PS I proteins do not accumulate to normal levels. Assembly of the peripheral PS I proteins PsaC,PsaD, PsaE, and PsaL is dependent on the presence of the PsaA and PsaB heterodimer core. The precursor form of PsaF may be inserted into the thylakoid membrane but is not processed to its mature form in the absence of PsaA and PsaB. The absence of PS I reaction centers has no apparent effect on Photosystem II (PS II) assembly and activity. Although the mutant exhibited somewhat greater fluorescence emission from phycocyanin, most of the light energy absorbed by phycobilisomes was efficiently transferred to the PS II reaction centers in the absence of the PS I. No light state transition could be detected in the psaAB::cat strain; in the absence of PS I, cells remain in state 1. Development of this relatively light-tolerant strain lacking PS I provides an important new tool for the genetic manipulation of PS I and further demonstrates the utility of Synechococcus sp. PCC 7002 for structural and functional analyses of the PS I reaction center.Abbreviations ATCC American type culture collection - Chl chlorophyll - DCMU 3-(3,4-dichlorophyl)-1,1-dimethylurea - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - HEPES N-[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic acid] - PCC Pasteur culture collection - PS I Photosystem I - PS II Photosystem II - SDS sodium dodecyl sulfate     

Photosynthetic acclimation of higher plants to growth in fluctuating light environments

This paper describes a study into the potential of plants to acclimate to light environments that fluctuate over time periods between 15 min and 3 h. Plants of Arabidopsis thaliana (L.) Heynh., Digitalis purpurea L. and Silene dioica (L.) Clairv. were grown at an irradiance 100 mol m^-2 s^-1. After 4–6 weeks, they were transferred to light regimes that fluctuated between 100 and either 475 or 810 mol m^-2 s^-1, in a regular cycle, for 7 days. Plants were shown, in most cases, to be able to undergo photosynthetic acclimation under such conditions, increasing maximum photosynthetic rate. The extent of acclimation varied between species. A more detailed study with S. dioica showed that this acclimation involved changes in both Rubisco protein and cytochrome f content, with only marginal changes in pigment content and composition. Acclimation to fluctuating light, at the protein level, did not fully reflect the acclimation to continuous high light - Rubisco protein increased more than would be expected from the mean irradiance, but less than expected from the high irradiance; cytochrome f increased when neither the mean nor the high irradiance would be expected to induce an increase.This revised version was published online in October 2005 with corrections to the Cover Date.     

Interchromosomal and intrachromosomal recombination in rad 18 mutants of Saccharomyces cerevisiae

Summary The frequency of intra- and interchromosomal recombination was determined in RAD18 and rad18 deletion and rad18-3 mutant strains. It was found that spontaneous interchromosomal recombination at trp5, his1, ade2, and MAT was elevated 10- to 70-fold in the rad18-3 and rad18 mutants as compared to the RAD ⁺ strains. On the other hand the frequencies of spontaneous intrachromosomal recombination for the his33, his35 and the his4C ^–, his4A ^– duplications and for heterothallic mating type switching were only marginally elevated in the rad18 deletion mutant, and recombination between ribosomal DNA repeats was only 2-fold elevated in the rad18-3 mutant. These differences may be due to a haploid versus diploid specific difference. However interchromosomal recombination was elevated 40-fold and intrachromosomal recombination was only marginally (1.5-fold) elevated in a diploid homozygous for rad18, arguing against a haploid versus diploid specific difference. Possible explanations for the difference in the elevated levels of intra- versus interchromosomal spontaneous recombination are discussed.     

Genetical studies on the heterocyst and nitrogen fixation of the blue-green alga Nostoc muscorum

Summary The short-trichome-forming, non-heterocystous and non-nitrogen-fixing (het ^– nif^–) mutant of the nitrogen-fixing blue-green alga Nostoc muscorum was isolated by N-methyl-N-nitro-N-nitrosoguanidine (NTG)-mutagenesis after penicillin enrichment technique and characterized. The mutant did not grow and fix nitrogen in combined-nitrogen-free medium while in nitrate-containing medium it grew well (K=0.112/day, G=64.27 h), although its growth was comparatively poor than the parent alga (K=0.128/day; G=56.14 h). The mutant was stable and both the het ^– and nif ^– characters reverted to wild type (het ⁺ nif⁺) with the reversion frequency of 2.62×10^-7.The het ^– nif^– mutant tolerated 0.5 g/ml of streptomycin sulphate on the agar medium and its streptomycin resistant mutant capable of growing in presence of 10g/ml of streptomycin was isolated spontaneously with a frequency of 1.45×10^-8. These streptomycin resistant isolates (het ^– nif^– str^R) resisted 100 g/ml of streptomycin sulphate on the agar medium and 200 g/ml in liquid medium. Spontaneous virus-resistant mutant of het ^– nif^– str^R was isolated with a mutation frequency of 4.02×10^-4.The data of genetic recombination experiments suggested that there is transfer of both het and nif genes to het ^– nif^– strain with the frequency of 2×10^-6 to 2×10^-5 simultaneously. There was increase in recombination frequency with increasing the incubation period. The virus-resistance marker is also transferred to the sensitive recipient.Abbreviations CFU colony forming units - C–N Chu-10 medium without combined nitrogen - C+N Chu-10 medium with 0.232 g/l calcium nitrate - G generation time - het heterocyst differentiating genes - K specific growth rate constant - MOI multiplicity of infection - nif nitrogen-fixing genes - NTG N-methyl-N-nitro-N-nitrosoguanidine - PFU plaque forming units - str ^R streptomycin resistance - str ^R streptomycin sensitive     

Functional analysis of the eyespot in Chlamydomonas reinhardtii mutant ey 627, mt −

The function of the eyespot in phototaxis of the flagellate green alga Chlamydomonas reinhardtii Dangeard was studied using quantitative reflection confocal laser scanning microscopy and photoelectric measurements. The reflective properties of the eyespot and the photoreceptor current of the C. reinhardtii eyespot mutant ey 627, mt ^– were compared with those of Chlamydomonas strains possessing a well-developed eyespot. Under growth conditions in which strongly disorganized eyespots were observed in the mutant by electron microscopy, there was a significant reduction in the reflection intensity of the eyespot and in the amplitude ratio (500440 nm) of photoreceptor currents induced by flashes of 500- and 440-nm light in non-oriented cells. Photoelectrical responses of pre-oriented cells revealed that the latter effect is caused by an altered directional sensitivity of the antenna complex, whereas the functional state of the photoreceptor pigment is not strongly affected in mutant cells. Both the reflection intensity and the amplitude ratio of photoreceptor currents increased to the level of reference strains under conditions supporting the development of a well-organized eyespot in the mutant. Furthermore, incubation of the mutant with high concentrations of all-trans-retinal (10 M), independent of whether carotenoid biosynthesis was inhibited or not, was found to increase the reflection intensity of the eyespot. An increase in the rate of photoorientation of the mutant occurred concomitant with the increase in the reflective properties of the mutant eyespot. These observations demonstrate the importance of an intact eyespot for interference reflection and absorption of phototactically active light, and thus for the directional sensitivity of the eyespot apparatus.Abbreviations HSM high-salt medium This study was supported by the Deutsche Forschungsgemeinschaft. O. A. Sineshchekov was supported by a Research Fellowship from the Alexander von Humboldt Foundation. The authors wish to thank U. Powalowski (Botanisches Institut, Universität zu Köln) for help with electron microscopy.     

Characteristics of DNA repair of a UV-sensitive mutant (radC) of Dictyostelium discoideum

Summary A radiation-sensitive mutant, TW8(radC), of Dictyostelium discoideum is more sensitive to ultraviolet light (UV) killing than the parental wild strain NC4(RAD ⁺), but is resistant to 4-nitroquinoline 1-oxide (4NQO) at almost the same level as NC4. In TW8 amoebae, single-strand breaks of DNA molecules were hardly detectable immediately after UV irradiation, and the removal of pyrimidine dimers was depressed during the postirradiation incubation when compared with that of NC4 amoebae. After treatment with 4NQO, however, single-strand breaks were detected in TW8 amoebae. The almost complete rejoining of these breaks was also detected after the removal of 4HAQO-adducts. The TW8 amoebae have an efficient repair capacity against DNA damage caused by 4NQO, MMS, MMC and MNNG but not UV.Abbreviations 4NQO 4-nitroquinoline 1-oxide - MMS methyl methanesulphonate - MMC mitomycin C - MNNG N-methyl-N-nitro-N-nitrosoguanidine     

Mutants of Rhizobium capable of fixing N2 in the free-living condition

Six mutant strains of Rhizobium were isolated after UV treatment which could exhibit nitrogenase activity in Burk's N-free medium without any supplement. The activity ranged between 99.5 and 113 nmol/mg cell dry weight and hour. Two of the parent strains belonged to soybean, and one each to mungbean and Sesbania sp. Both the parent and mutant strains exhibited nitrogenase activity in CS 7 medium. One of the mutants retained its capacity to produce nodules on soybean roots.List of Abbreviations C.D. Critical difference - EMS ethylmethane sulphonate - NTG N-methyl-N-nitro, N-nitrosoguanidine     

Inactivation of the water-oxidizing enzyme in manganese stabilizing protein-free mutant cells of the cyanobacteria Synechococcus PCC7942 and Synechocystic PCC6803 during dark incubation and conditions leading to photoactivation

The previously constructed MSP (manganese stabilizing protein-psbO gene product)-free mutant of Synechococcus PCC7942 (Bockholt R, Masepohl B and Pistorius E K (1991) FEBS Lett 294: 59–63) and a newly constructed MSP-free mutant of Synechocystis PCC6803 were investigated with respect to the inactivation of the water-oxidizing enzyme during dark incubation. O₂ evolution in the MSP-free mutant cells, when measured with a sequence of short saturating light flashes, was practically zero after an extended dark adaptation, while O₂ evolution in the corresponding wild type cells remained nearly constant. It could be shown that this inactivation could be reversed by photoactivation. With isolated thylakoid membranes from the MSP-free mutant of PCC7942, it could be demonstrated that photoactivation required illumination in the presence of Mn²⁺ and Ca²⁺, while Cl^– addition was not required under our experimental conditions. Moreover, an extended analysis of the kinetic properties of the water-oxidizing enzyme (kinetics of the S₃(S₄)S₀ transition, S-state distribution, deactivation kinetics) in wild type and mutant cells of Synechococcus PCC7942 and Synechocystis PCC6803 was performed, and the events possibly leading to the reversible inactivation of the water-oxidizing enzyme in the mutant cells are discussed. We could also show that the water-oxidizing enzyme in the MSP-free mutant cells is more sensitive to inhibition by added NH₄Cl-suggesting that NH₃ might be a physiological inhibitor of the water oxidizing enzyme in the absence of MSP.Abbreviations Chl chlorophyll - DCBQ 2,6-Dichloro-p-benzoquinone - MSP manganese stabilizing protein (psbO gene product) - PS II Photosystem II - WOE water oxidizing enzyme - WT wild type This paper is dedicated to Prof. Dr. Bernard Axelrod on the occasion of his 80th birthday     

Isolation and identification of blue light-induced growth inhibitor from light-grown <Emphasis Type="Italic">Arabidopsis</Emphasis> shoots

Phototropic stimulation of dark-grown hypocotyls of Arabidopsis thaliana increased a growth inhibitor in the wild-type but not in the non-phototropic nph3-101 mutant. From light-grown wild-type shoots the inhibitor was isolated and identified as indole-3-acetonitrile (IAN) from its ¹H NMR spectrum. The content of endogenous IAN in the hypocotyls of wild-type and mutant unilaterally exposed to blue light was determined using a physicochemical assay. The IAN concentration (28 M) in the phototropically stimulated wild-type hypocotyls was about three times larger than in the dark control. However, its content in the mutant hypocotyls did not change. IAN inhibited the hypocotyl growth of the nph3-101 to the same extent as in the wild-type at concentrations higher than 10 M. These results suggest that IAN plays a role in the phototropism of Arabidopsis thaliana hypocotyls.     

Phototactic responses in Haematococcus lacustris and its modification by light intensity and the carotenoid biosynthesis inhibitor Norflurazon

At fluence rates below 45 W· m^-2 cells of the flagellate stage of Haematococcus lacustris react only positively phototactically with a rather high degree of orientation (indicated by r values up to 0.66 with the Rayleigh test). The directedness of orientation decreases with decreasing irradiance. The degree of directedness of the phototactic response depends on the intensity of preirradiation: Low light intensity applied after strong light application results in a dark reaction (low r values), low light given after darkness stimulates a rather high degree of directedness of positive phototaxis. Weak blue light (=483 nm; 0.4 W · m^-2) stimulates positive phototactic response, whereas comparable red light (=658 nm; 0.5 W · m^-2) does not.Cells which were grown in a medium containing 10^-4 M Norflurazon (effective in inhibition of carotenoid biosynthesis) although maintaining motility completely lose the ability to react positively phototactically. The possible role of carotenoids in the phototactic orientation is discussed.     

Changes of amino acid sequence in PEST-like area and QEEET motif affect degradation rate of D1 polypeptide in photosystem II

The degradation rate of the D1 polypeptide was measured in threeSynechocystis PCC 6803 mutantsin vivo. Mutations were introduced into a putative cleavage area of the D1 polypeptide (QEEET motif) and into the PEST-like area. PEST sequences are often found in proteins with a high turnover rate. The QEEET-motif mutants are CA1 [(E242-E244);Q241H] and E243K, and the third mutation, E229D, was directed to the PEST-like area. During high-light illumination (1500 mol photons m^-2s^-1) that induced photoinhibition of photosystem II (PSII), the half-life time of the D1 polypeptide in mutant E229D (t _1/2=35 min) was about twice as long as in AR (control strain) cells (t _1/2=19 min). In growth light (40 mol photons m^-2s^-1), the degradation rate of the D1 polypeptide in E229D and AR strains was the same (t _1/25 h). In growth light the D1 polypeptide was degraded faster in both QEEET-motif mutants than in the AR strain, but in photoinhibitory light the degradation rates were similar. According to these results, the highly conservative QEEET motif as such is not required for the proteolytic cut of the D1 polypeptide, but it does affect the rate of degradation. No simple correlation existed between the degradation rate of the D1 polypeptide and the susceptibility of PSII to photoinhibition in mutant and AR cells under our experimental conditions.     

Light-path length and population density in photoacclimation of Nannochloropsis sp. (Eustigmatophyceae)

Photoacclimation in the marine eustigmatophyte Nannochlropsis sp., used extensively as a food chaincomponent in aquaculture, was studied both in thelaboratory and outdoors. Cell-chlorophyll andcarotenoids were used as markers to assessphotoacclimation to strong light, as well as todecreasing growth irradiance due to cellproliferation. Focusing on practical aspects involvedin mass cultivation, three different approaches wereused as follows: (a) cultures initially exposed to lowlight (150 mol photon m^-2 s^-1) thentransferred to strong light (1000 to 3000 molphoton m^-2 s^-1); (b) initially low celldensity cultures grown in reactors of differentlight-paths, exposed to strong PFD, in the laboratoryand outdoors; (c) initially low or high cell densitycultures exposed to strong light. As has already beenestablished in many reports, cell-chlorophyllrepresented a sensitive parameter in assessing cellresponse to changes in the intensity of the lightsource as well as to modifications in the light regimeto which the cells were exposed. Cell-chlorophyllconcentration sharply decreased initially upontransferring the culture from low PFD cell^-1 tohigh PFD cell^-1 due to either culture dilution(i.e. decrease in cell density and mutual shading) orto an increase in PFD. After some 7 days ofphotoacclimating to 2000 and 3000 mol photonm^-2 s^-1, chlorophyll a content began to riseat a much faster rate than cell number, which alsoincreased in response to the higher irradiance.Cell-chlorophyll in the culture exposed to 2000mol photon m^-2 s^-1 increased afteracclimation earlier and at a faster rate than in theculture exposed to 3000 mol photon m^-2s^-1, indicating the later irradiance affected astronger stress. The length of the reactor's lightpath exerted a decisive effect on cell response tostrong light through its influence on the light regimein the culture. Upon a sharp increase in PFD,carotenoids in the 1-cm reactor increased in muchhigher rate than chlorophyll, compared with the 3-cmlight path reactors. This marked difference in cellresponse to a shift-up in light was attributed to thevast variations in the light regime associated withdifferences in the length of the light path and areal density. Growth oflow cell density cultures ceased temporarily upontransfer to strong light, in contrast with high celldensity cultures transferred to strong light, whichcontinued growth without a lag.     

Genetic mapping by means of protoplast fusion in Bacillus subtilis

Summary A new mapping method involving protoplast fusion in Bacillus subtilis is described. Protoplasts from an isogenic standard marker strain containing purA and from a strain containing both purB and the marker, x, to be mapped were fused with polyethylene glycol, and purA ⁺ purB ⁺ fusants were selected. After isolation of single colonies and determination of unselected markers, marker x was mapped between two standard markers. This method was fully applicable to PBS1-resistant strains (e.g., lyt strains). The results obtained by protoplast fusion, conventional transformation and/or lysed protoplast transformation indicated that a lyt strain, Ni15, contained two new autolysin-minus mutations (lyt-151 and lyt-152). The properties of lyt-15 are also discussed.Abbreviations NTG N-methyl-N-nitro-N-nitrosoguanidine - SMM 0.5 M sucrose, 0.02 M MgCl₂, 0.02 M maleate buffer, pH 6.5