Non-inducing Medium Research Articles

Programmed cell death in Xanthomonas axonopodis pv. glycines is associated with modulation of gene expression resulting in altered states of motility, biofilm and virulence

One of the foremost report of apoptosis-like programmed cell death (PCD) came from Xanthomonas axonopodis pv. glycines (Xag), which displayed rapid post-exponential cell death in PCD inducing media (PIM) but not in a non-inducing media (PNIM). The current study aims to decipher for the first time, the advantages of the existence of PCD in this phytopathogenic microorganism. Analysis of RNA-seq under inducing and non-inducing conditions, revealed differential expression of a number of genes related to key physiology of Xag, such as, motility, xanthan biosynthesis and export as well as virulence. A PCD negative mutant Xag M42 displayed diminished virulence and a contrasting transcriptome pattern. In vitro experiments revealed that under PCD inducing condition, Xag produced negligible xanthan gum as well as extracellular amylase, displayed enhanced swarming motility, released copious e-DNA and formed scanty biofilm. Lack of ‘diffusible signalling factor’ production was eliminated as possible reason for PCD-induction. Altogether, it appears that, in planta existence of the pathogen metabolically resembles PNIM, and on being transferred to PIM, the cells experience oxidative stress and circumvents it by adopting PCD as an altruistic response. Survival of the remaining population is encouraged by upregulating motility, detachment from the fragile biofilm to achieve dispersal.

Characterization of the Multiple Domains of Pex30 Involved in Subcellular Localization of the Protein and Regulation of Peroxisome Number.

Pex30 is a peroxisomal protein whose role in peroxisome biogenesis via the endoplasmic reticulum has been established. It is a 58 KDa multi-domain protein that facilitates contact site formation between various organelles. The present study aimed to investigate the role of various domains of the protein in its sub-cellular localization and regulation of peroxisome number. For this, we created six truncations of the protein (1-87, 1-250, 1-352, 88-523, 251-523 and 353-523) and tagged GFP at the C-terminus. Biochemical methods and fluorescence microscopy were used to characterize the effect of truncation on expression and localization of the protein. Quantitative analysis was performed to determine the effect of truncation on peroxisome number in these cells. Expression of the truncated variants in cells lacking PEX30 did not cause any effect on cell growth. Interestingly, variable expression and localization of the truncated variants in both peroxisome-inducing and non-inducing medium was observed. Truncated variants depicted different distribution patterns such as punctate, reticulate and cytosolic fluorescence. Interestingly, lack of the complete dysferlin domain or C-Dysf resulted in increased peroxisome number similar to as reported for cells lacking Pex30. No contribution of this domain in the reticulate distribution of the proteins was also observed. Our results show an interesting role for the various domains of Pex30 in localization and regulation of peroxisome number.

Increased peroxisome proliferation is associated with early yeast replicative ageing.

Peroxisomes are single membrane-bound organelles ubiquitously present in several cell types and are associated with cell and tissue-specific functions. Their role in cellular ageing is under investigation in various model systems. Metabolism of cellular reactive oxygen species is a universal function performed by these organelles. In this study, we investigated alterations in peroxisome number upon early replicative ageing of yeast cells. Increase in the number of peroxisomes in replicatively aged mother cells of wild-type yeast was observed when cultured in both peroxisome-inducing and non-inducing medium. Further, we investigated if this increase in peroxisome number in replicatively aged cells is due to enhanced peroxisome proliferation. For this, the number of peroxisomes in replicatively aged mother cells of pex11, pex25 and pex11pex25 was analysed. Increased percentage of aged cells was observed in pex25 and pex11pex25 cells cultured in peroxisome-inducing oleic acid medium. Interestingly, when cultured in oleic acid, young mother cells devoid of Pex11 showed reduced peroxisome proliferation compared to old mother cells. Induced activity of the antioxidant enzyme catalase and reduced accumulation of reactive oxygen species were reported in all studied strains when cultured in oleic acid medium. Further, our data also suggest that replicatively aged cells with increased peroxisome number also display mitochondrial dysfunction and fragmentation in all the strains studied. In conclusion, our data suggests a correlation between increase in peroxisome number and replicative age of yeast cells and interestingly this increase seems to be partly dependent on the fission proteins.

Deferred control of ammonium cross-feeding in a N2-fixing bacterium-microalga artificial consortium.

There is an increasing interest in the use of N2-fixing bacteria for the sustainable biofertilization of crops. Genetically-optimized bacteria for ammonium release have an improved biofertilization capacity. Some of these strains also cross-feed ammonium into microalgae raising additional concerns on their sustainable use in agriculture due to the potential risk of producing a higher and longer-lasting eutrophication problem than synthetic N-fertilizers. Here we studied the dynamic algal cross-feeding properties of a genetically-modified Azotobacter vinelandii strain which can be tuned to over-accumulate different levels of glutamine synthetase (GS, EC 6.3.1.20) under the control of an exogenous inducer. After switching cells overaccumulating GS into a noninducing medium, they proliferated for several generations at the expense of the previously accumulated GS. Further dilution of GS by cell division slowed-down growth, promoted ammonium-excretion and cross-fed algae. The final bacterial population, and timing and magnitude of algal N-biofertlization was finely tuned in a deferred manner. This tuning was in accordance with the intensity of the previous induction of GS accumulation in the cells. This bacterial population behavior could be maintained up to about 15 bacterial cell generations, until faster-growing and nonammonium excreting cells arose at an apparent high frequency. Further improvements of this genetic engineering strategy might help to align efficiency of N-biofertilizers and safe use in an open environment. KEY POINTS: • Ammonium-excreting bacteria are potential eutrophication agents • GS-dependent deferred control of bacterial growth and ammonium release • Strong but transient ammonium cross-feeding of microalgae.

Periplasm-enriched fractions from Xanthomonas citri subsp. citri type A and X. fuscans subsp. aurantifolii type B present distinct proteomic profiles under in vitro pathogenicity induction.

The causative agent of Asiatic citrus canker, the Gram-negative bacterium Xanthomonas citri subsp. citri (XAC), produces more severe symptoms and attacks a larger number of citric hosts than Xanthomonas fuscans subsp. aurantifolii XauB and XauC, the causative agents of cancrosis, a milder form of the disease. Here we report a comparative proteomic analysis of periplasmic-enriched fractions of XAC and XauB in XAM-M, a pathogenicity- inducing culture medium, for identification of differential proteins. Proteins were resolved by two-dimensional electrophoresis combined with liquid chromatography-mass spectrometry. Among the 12 proteins identified from the 4 unique spots from XAC in XAM-M (p<0.05) were phosphoglucomutase (PGM), enolase, xylose isomerase (XI), transglycosylase, NAD(P)H-dependent glycerol 3-phosphate dehydrogenase, succinyl-CoA synthetase β subunit, 6-phosphogluconate dehydrogenase, and conserved hypothetical proteins XAC0901 and XAC0223; most of them were not detected as differential for XAC when both bacteria were grown in NB medium, a pathogenicity non-inducing medium. XauB showed a very different profile from XAC in XAM-M, presenting 29 unique spots containing proteins related to a great diversity of metabolic pathways. Preponderant expression of PGM and XI in XAC was validated by Western Blot analysis in the periplasmic-enriched fractions of both bacteria. This work shows remarkable differences between the periplasmic-enriched proteomes of XAC and XauB, bacteria that cause symptoms with distinct degrees of severity during citrus infection. The results suggest that some proteins identified in XAC can have an important role in XAC pathogenicity.

Expression of the Fusarium graminearum terpenome and involvement of the endoplasmic reticulum-derived toxisome

The sesquiterpenoid deoxynivalenol (DON) is an important trichothecene mycotoxin produced by the cereal pathogen Fusarium graminearum. DON is synthesized in specialized subcellular structures called toxisomes. The first step in DON synthesis is catalyzed by the sesquiterpene synthase (STS), Tri5 (trichodiene synthase), resulting in the cyclization of farnesyl diphosphate (FPP) to produce the sesquiterpene trichodiene. Tri5 is one of eight putative STSs in the F. graminearum genome. To better understand the F. graminearum terpenome, the volatile and soluble fractions of fungal cultures were sampled. Stringent regulation of sesquiterpene accumulation was observed. When grown in trichothecene induction medium, the fungus produces trichothecenes as well as several volatile non-trichothecene related sesquiterpenes, whereas no volatile terpenes were detected when grown in non-inducing medium. Surprisingly, a Δtri5 deletion strain grown in inducing conditions not only ceased accumulation of trichothecenes, but also failed to produce the non-trichothecene related sesquiterpenes. To test whether Tri5 from F. graminearum may be a promiscuous STS directly producing all observed sesquiterpenes, Tri5 was cloned and expressed in E. coli and shown to produce primarily trichodiene in addition to minor, related cyclization products. Therefore, while Tri5 expression in F. graminearum is necessary for non-trichothecene sesquiterpene biosynthesis, direct catalysis by Tri5 does not explain the sesquiterpene deficient phenotype observed in the Δtri5 strain. To test whether Tri5 protein, separate from its enzymatic activity, may be required for non-trichothecene synthesis, the Tri5 locus was replaced with an enzymatically inactive, but structurally unaffected tri5N225D S229T allele. This allele restores non-trichothecene synthesis but not trichothecene synthesis. The tri5N225D S229T allele also restores toxisome structure which is lacking in the Δtri5 deletion strain. Our results indicate that the Tri5 protein, but not its enzymatic activity, is also required for the synthesis of non-trichothecene related sesquiterpenes and the formation of toxisomes. Toxisomes thus not only may be important for DON synthesis, but also for the synthesis of other sesquiterpene mycotoxins such as culmorin by F. graminearum.

Biosynthesis of Antibiotic Leucinostatins in Bio-control Fungus Purpureocillium lilacinum and Their Inhibition on Phytophthora Revealed by Genome Mining.

Purpureocillium lilacinum of Ophiocordycipitaceae is one of the most promising and commercialized agents for controlling plant parasitic nematodes, as well as other insects and plant pathogens. However, how the fungus functions at the molecular level remains unknown. Here, we sequenced two isolates (PLBJ-1 and PLFJ-1) of P. lilacinum from different places Beijing and Fujian. Genomic analysis showed high synteny of the two isolates, and the phylogenetic analysis indicated they were most related to the insect pathogen Tolypocladium inflatum. A comparison with other species revealed that this fungus was enriched in carbohydrate-active enzymes (CAZymes), proteases and pathogenesis related genes. Whole genome search revealed a rich repertoire of secondary metabolites (SMs) encoding genes. The non-ribosomal peptide synthetase LcsA, which is comprised of ten C-A-PCP modules, was identified as the core biosynthetic gene of lipopeptide leucinostatins, which was specific to P. lilacinum and T. ophioglossoides, as confirmed by phylogenetic analysis. Furthermore, gene expression level was analyzed when PLBJ-1 was grown in leucinostatin-inducing and non-inducing medium, and 20 genes involved in the biosynthesis of leucionostatins were identified. Disruption mutants allowed us to propose a putative biosynthetic pathway of leucinostatin A. Moreover, overexpression of the transcription factor lcsF increased the production (1.5-fold) of leucinostatins A and B compared to wild type. Bioassays explored a new bioactivity of leucinostatins and P. lilacinum: inhibiting the growth of Phytophthora infestans and P. capsici. These results contribute to our understanding of the biosynthetic mechanism of leucinostatins and may allow us to utilize P. lilacinum better as bio-control agent.

Genetically engineeredFusariumas a tool to evaluate the effects of environmental factors on initiation of trichothecene biosynthesis

Fusarium graminearum was engineered for expression of enhanced green fluorescent protein gene (egfp) as a reporter regulated in a manner similar to Tri5, a key pathway gene in trichothecene biosynthesis. Using the transgenic fungus, it was found that the reporter gene was induced to express in aerial hyphae developed on trichothecene noninducing medium YG solidified by agar. Unexpectedly, the transcriptional activation of egfp was markedly suppressed by adding NaCl that does not significantly affect fungal growth. As suggested by these findings, wild-type F. graminearum that formed aerial hyphae on YG agar plates produced trichothecenes and the production was effectively suppressed by adding 1% NaCl to the agar. To evaluate the effects of abiotic stress on the expression of trichothecene biosynthesis (Tri) genes, a sensitive plate assay was established using GYEP medium (which very weakly induces trichothecene production) solidified with gellan gum. Using this assay, triazole fungicides were shown to cause transcriptional activation of egfp at sublethal concentrations. Indeed, trichothecene production significantly increased when F. graminearum was grown in rice medium (which moderately induces trichothecene) amended with low doses of tebuconazole. The real-time monitoring system described here may help predict the risks of trichothecene contamination by the fungus under various environmental conditions.

Characterization of a flocculation-like phenotype in Cryptococcus neoformans and its effects on pathogenesis

We investigated the phenomenon of cell-cell aggregation (flocculation) in a serotype D strain of Cryptococcus neoformans (ATCC 24067, isolate RC-2). Cell aggregation into clumps of 5-40 cells (clump+ cells) occurred during the early log phase and disappeared in the beginning of the stationary phase (clump- cells). The cell aggregation phenomenon was medium dependent. Clump+ cells could be dispersed by either vortexing or proteinase K digestion. Most importantly, the transient change in cellular phenotype changed several important host-pathogen interactions. Adherence of clump+ cells to murine macrophage-like cells J774.16 was significantly (P < 0.001) enhanced compared with adherence of clump- cells. Furthermore, complement-mediated phagocytosis efficacy of dispersed clump+ cells was significantly higher (P < 0.001) compared with clump- cells. Similar findings were documented with an in vivo phagocytosis assay. Infection of mice with a low inoculum (10(4)) of clump+ cells resulted in lower fungal burden when compared with mice infected with clump- cells. Accordingly, mice infected with clump+ cells survived significantly longer than mice infected with clump- cells. These results indicate that the cellular phenotype undergoes significant changes that result in a transient flocculation-like phenotype. We hypothesize that this cell-cell aggregation is the result of changes in protein content in the polysaccharide capsule. We conclude from our data that the change in cellular phenotype has a dramatic effect on cell adherence, and on complement-mediated phagocytosis, both of which can affect the pathogenesis of the disease in the host. Our results underscore the complexity of studies that investigate host pathogen interactions and may explain differences and inconsistencies observed in in vitro and in vivo assays.

Regulation of IκB Kinase (IKK) Complex by IKKγ-dependent Phosphorylation of the T-loop and C Terminus of IKKβ

Regulation of IκB Kinase (IKK) Complex by IKKγ-dependent Phosphorylation of the T-loop and C Terminus of IKKβ

The polysaccharide capsule of the pathogenic fungus Cryptococcus neoformans enlarges by distal growth and is rearranged during budding

The capsule of Cryptococcus neoformans can undergo dramatic enlargement, a phenomenon associated with virulence. A prior study that used Ab to the capsule as a marker for older capsular material concluded that capsule growth involved the intermixing of new and old capsular material with displacement of older capsular polysaccharide towards the surface. Here we have revisited that question using complement (C), which binds to capsular polysaccharide covalently, and cannot redistribute by dissociation and binding at different sites. The experimental approach involved binding of C to cells with small capsules, inducing capsule growth, and following the location of C relative to the cell wall as the capsule enlarged. C remained close to the cell wall during capsule growth, indicating that capsule enlargement occurred by addition of new polysaccharide near the capsule edge. This conclusion was confirmed by an independent method that employed radioactive metabolic labelling of newly synthesized capsule with 3H-mannose followed by gradual capsular stripping with gamma-radiation. Capsule growth proceeded to a certain size, which was a function of cell size, and was not degraded when the cells were transferred to a non-inducing medium. During budding, an opening appeared in the capsule of the mother cell that permitted the nascent bud to separate. Scanning EM suggested that a physical separation formed between the capsules of the mother and daughter cells during budding, which may avoid mixture between both capsules. Our results indicate that C. neoformans capsular enlargement also occurs by apical growth and that budding results in capsular rearrangements.

Multiple components and induction mechanism of the chitinolytic system of the hyperthermophilic archaeon Thermococcus chitonophagus.

Thermococcus chitonophagus produces several, cellular and extracellular chitinolytic enzymes following induction with various types of chitin and chitin oligomers, as well as cellulose. Factors affecting the anaerobic culture of this archaeon, such as optimal temperature, agitation speed and type of chitin, were investigated. A series of chitinases, co-isolated with the major, cell membrane-associated endochitinase (Chi70), and a periplasmic chitobiase (Chi90) were subsequently isolated. In addition, a distinct chitinolytic activity was detected in the culture supernatant and partially purified. This enzyme exhibited an apparent molecular mass of 50 kDa (Chi50) and was optimally active at 80 degrees C and pH 6.0. Chi50 was classified as an exochitinase based on its ability to release chitobiose as the exclusive hydrolysis product of colloidal chitin. A multi-component enzymatic apparatus, consisting of an extracellular exochitinase (Chi50), a periplasmic chitobiase (Chi90) and at least one cell-membrane-anchored endochitinase (Chi70), seems to be sufficient for effective synergistic in vivo degradation of chitin. Induction with chitin stimulates the coordinated expression of a combination of chitinolytic enzymes exhibiting different specificities for polymeric chitin and its degradation products. Among all investigated potential inducers and nutrient substrates, colloidal chitin was the strongest inducer of chitinase synthesis, whereas the highest growth rate was obtained following the addition of yeast extract and/or peptone to the minimal, mineralic culture medium in the absence of chitin. In rich medium, chitin monomer acted as a repressor of total chitinolytic activity, indicating the presence of a negative feedback regulatory mechanism. Despite the undisputable fact that the multi-component chitinolytic system of this archaeon is strongly induced by chitin, it is clear that, even in the absence of any chitinous substrates, there is low-level, basal, constitutive production of chitinolytic enzymes, which can be attributed to the presence of traces of chito-oligosaccharides and other structurally related molecules (in the undefined, rich, non-inducing medium) that act as potential inducers of chitinolytic activity. The low, basal and constitutive levels of chitinase gene expression may be sufficient to initiate chitin degradation and to release soluble oligomers, which, in turn, induce chitinase synthesis.

Characterization of the hrp pathogenicity cluster of Erwinia carotovora subsp. carotovora: high basal level expression in a mutant is associated with reduced virulence.

Extracellularly targeted proteins are crucial for virulence of gram-negative phytopathogenic bacteria. Erwinia carotovora subsp. carotovora employs the so-called type II (GSP) pathway to secrete a number of pectinases and cellulases, which cause the typical tissue maceration symptoms of soft-rot disease. The type III (hrp) pathway is the major virulence determinant in the genera Pseudomonas, Ralstonia and Xanthomonas, and in non-macerating species of Erwinia. The hrp cluster was recently partially characterized from E. carotovora sp. carotovora, and shown to affect virulence during early stages of infection. Here we have isolated and characterized 15 hrp genes comprising the remaining part of the cluster. The genes hrpL, hrpXY and hrpS were deduced to be transcribed as separate units, whereas the 11 remaining genes from hrpJ to hrcU form a single large operon. The hrpX gene, which codes for the sensory kinase of the two-component regulatory locus hrpXY was insertionally inactivated by placing a transposon (entranceposon) in the gene. The resulting mutant bacterium expresses the hrp genes at high basal level even in a non-inducing medium. This relative overexpression was shown to be due to the hrpX::entranceposon insertion causing enhanced transcription of the downstream hrpY gene. The hrpX(-)-hrpYC mutant bacterium exhibited a slower growth rate and the appearance of disease symptoms in infected Arabidopsis plants was delayed, as compared to the wild-type strain. The need for hrp gene expression for virulence has been documented in both non-macerating plant pathogens and in soft-rotting Erwinia sp. but this is the first demonstration that high basal-level expression of hrp -regulated genes may actually have a negative impact on disease progress in a susceptible host plant.

Transformants of Metarhizium anisopliae sf. anisopliae overexpressing chitinase from Metarhizium anisopliae sf. acridum show early induction of native chitinase but are not altered in pathogenicity to Manduca sexta.

Extracellular chitinase activity has been implicated in the pathogenesis of several fungal infections. Following induction with chitin, the insect pathogens Metarhizium anisopliae sf. acridum ARSEF strain 324 and Metarhizium anisopliae sf. anisopliae ARSEF strain 2575 secrete 44-kDa basic and acidic isoforms of endochitinase, respectively. The gene from strain 324 (Chit1) was cloned and inserted into the genome of strain 2575 under the control of Aspergillus regulatory elements such that transgenic 2575 (2575-Chit(+)) expressed CHIT1 in a noninducing medium (i.e., not containing chitin). Isoelectric focusing followed by a zymogram technique revealed that neither wild-type 2575 nor 2575-Chit(+) produced significant amounts of the native 2575 acidic chitinase in a noninducing medium. However, in a chitin-containing medium, 2575-Chit(+) produced the native chitinase earlier than strain 2575, soon after secretion of CHIT1. We hypothesize that this is due to the production of soluble inducers following chitin hydrolysis by CHIT1 and that M. anisopliae uses enzymes expressed at low levels to sense the nature of the polymeric nutrient present in the immediate environment. However, the chitinase overproducers did not show altered virulence to caterpillars (Manduca sexta) compared to the wild-type fungus, suggesting that wild-type levels of chitinase are not limiting for cuticle penetration.

Extracellular polysaccharide composition of Azospirillum brasilense and its relation with cell aggregation

The exopolysaccharide (EPS) and capsular polysaccharide (CPS) composition of four Azospirillum brasilense strains differing in their aggregation capacity was analyzed by high performance anion exchange chromatography. When growing the different strains in an aggregation inducing medium containing a high carbon:nitrogen (C:N) ratio, both EPS and CPS showed a positive correlation between aggregation and the relative amount of arabinose. Arabinose was not detected in polysaccharides from Sp72002, a pleiotrophic Tn5 mutant strain impaired in aggregation. Arabinose was also not detected in extracellular polysaccharides of bacteria grown in a low C:N ratio, non-inducing aggregation medium, with exception for a relatively small amount found in the CPS of FAJ0204, a super-aggregating mutant strain. The only monosaccharides able to significantly inhibit aggregation at low sugar concentration when tested in a bioassay were arabinose (at a higher extent) and galactose. The possibility that residues of arabinose present in the extracellular polysaccharides are involved in the aggregation of A. brasilense is discussed.

Extracellular polysaccharide composition of Azospirillum brasilense and its relation with cell aggregation.

The exopolysaccharide (EPS) and capsular polysaccharide (CPS) composition of four Azospirillum brasilense strains differing in their aggregation capacity was analyzed by high performance anion exchange chromatography. When growing the different strains in an aggregation inducing medium containing a high carbon:nitrogen (C:N) ratio, both EPS and CPS showed a positive correlation between aggregation and the relative amount of arabinose. Arabinose was not detected in polysaccharides from Sp72002, a pleiotrophic Tn5 mutant strain impaired in aggregation. Arabinose was also not detected in extracellular polysaccharides of bacteria grown in a low C:N ratio, non-inducing aggregation medium, with exception for a relatively small amount found in the CPS of FAJ0204, a super-aggregating mutant strain. The only monosaccharides able to significantly inhibit aggregation at low sugar concentration when tested in a bioassay were arabinose (at a higher extent) and galactose. The possibility that residues of arabinose present in the extracellular polysaccharides are involved in the aggregation of A. brasilense is discussed.

A conidial trap-forming mutant of Arthrobotrys oligospora

Conidial traps (Ct) in Arthrobotrys oligospora are adhesive structures formed directly on germination of conidia, without an intermediate hyphal phase. A conidial trap-producing mutant was isolated by a procedure analogous to that used for microcycle conidiation. This Ct-strain produced strict conidial traps in abundance on conidia when still on upright conidiophores. The phenotypic stability of the Ct-strain lasted over more than 20 transfers on trap-inducing media. Growth on non-inducing media (5 transfers) and subsequent inoculation on trap-inducing medium again showed traps on conidia on upright conidiophores. The conidial traps attached to nematodes before invasion and digestion. At the subcellular level the Ct were characterized by the presence of many electron-dense microbodies. In addition, in the Ct-strain normal trap formation on the mycelium was considerably increased compared to the parent strain. This high trap-forming ability will make it a useful tool both in rhizosphere studies and in biochemical/molecular investigations.

Cell Wall Composition and Morphogenic Response in Callus Derived from Protoplasts of two Fibre flax (Linum usitatissimum L.) Genotypes

Summary Cotyledonary protoplasts of two French fibre flax ( Linum usitatissimum ) cultivars (Ariane and Viking) were isolated and cultured. The optimal conditions for high yield were from 4-5 day old seedlings, where 6-6.5x10 6 and 6.7-7.4x10 6 protoplasts were released from cv. Ariane and cv. Viking respectively. Culture in glutamine (20mM) containing medium led to first divisions after 2 days with an efficiency of 19% or 21.9% (cv. Ariane) and 49.3% or 44% (cv. Viking) of divided cells, when plating densities were 10 5 versus 5 x 10 4 protoplasts per mL. Rhizogenesis occurred on protoplast derived calli at high frequency 4 weeks after transfer onto solid medium supplemented with 0.5 μM 2.4 dichlorophenoxyacetic acid. Variations of cell wall sugar composition during culture on inducing and non-inducing media expressed some significant differences. The percentage of galacturonic acid increased on non-inducing medium, but remained constant on root-inducing medium. Glucose presented a peak of accumulation concomitant of the emergence of the root meristems 4 weeks after transfer onto 2,4-D containing medium. On the contrary galactose and arabinose variations were not characteristic of the rhizogenic process.

Cooperative regulation of cell proliferation by calcium and calmodulin in Aspergillus nidulans.

Calcium and calmodulin have been widely implicated in the control of cell proliferation. We have created a strain of the genetically tractable filamentous fungus, Aspergillus nidulans, that is conditional for calmodulin expression. This was accomplished by replacing the unique endogenous calmodulin gene with one regulated by the inducible alcohol dehydrogenase (alcA) gene promoter by homologous recombination. This strain cannot grow when the cells are incubated in medium containing a carbon source that represses the alcA promoter. Characterization of the arrested cells shows that 83% are blocked in the G2 phase of the cell cycle. The block is due to very low levels of calmodulin and is fully reversible upon changing to medium that contains an inducer of the alcA promoter. The rate of cell proliferation in this strain is dependent upon both the intracellular calmodulin and extracellular Ca2+ concentrations. Raising the calmodulin concentration by inducing the alcA promoter not only causes the cells to enter the proliferative cycle more quickly and to grow faster, but also decreases the concentration of extracellular Ca2+ required to support growth by 10-fold, as compared with cells grown in noninducing medium. Thus both the intracellular calmodulin and extracellular Ca2+ concentrations are important and interactive factors in regulating the nuclear division cycle of Aspergillus nidulans.

Induction of Methanethiol Production by Brevibacterium linens CNRZ 918

SUMMARY: A non-inducing medium (NID) was defined for studying the induction of methanethiol production by Brevibacterium linens CNRZ 918. The lowest l-methionine concentration capable of inducing maximal methanethiol production by the cells was 7 mM. The peptides l-Ala-l-Met and l-Met-l-Ala induced greater methanethiol production than free l-methionine. D-Methionine, l-cysteine, S-methyl-l-cysteine and l-ethionine were poor inducers. Culture temperature affected the duration of induction. An Na+ concentration of 1 M in the culture medium led to maximal methanethiol production capacity of both cells and cell extracts. l-Methionine and l-ethionine were the best substrates for the crude soluble cells extract (with release of methanethiol and ethanethiol respectively). Neither the derivatives of l-methionine that acted as inducers, nor D-methionine, were substrates for demethiolation. Demethiolation activity of the crude extract was thermolabile, not stimulated by Na+ and strongly inhibited by Zn2+, Mn2+ and Cu2+. The shortest generation time obtained for B. linens CNRZ 918 in NID medium + l-methionine was 4 h at 26 °C. Only coccoid forms were present when the culture temperature was 30 °C. The presence of l-methionine in the medium favoured their appearance. The strain grew best in the presence of 1% NaCl but tolerated concentrations up to 15%. The induction of methanethiol production was due to the induction of l-methionine-γ-demethiolase. The level of induction was probably related to the intracellular concentration of l-methionine. The transport system of l-methionine by B. linens CNRZ 918 was constitutive and Na+ dependent.