Effect of changes in mineral composition and growth temperature on filament length and flagellation in the Archaeon Methanospirillum hungatei

Methanospirillum hungatei strains GP1 and JF1 when cultivated at 37°C in JMA medium grew as motile single cells or short chains of cells (typically 10–30 m long). When M. hungatei was grown in low Ca²⁺ concentrations or with the divalent cation chelator EDTA, the organism grew as long non-flagellated filaments (up to 900 m long). The two strains had different thresholds of calcium concentrations for long filament formation (<0.25 mM for GP1 and <0.15 mM for JF1) as well as different minimal Ca²⁺ requirements for growth. Both strains produced long, almost straight, filaments at Ca²⁺ concentrations near the minimum required for growth. At suboptimal growth temperatures the organisms still grew as short filaments but no longer possessed flagella. Western blot analysis indicated that flagellin monomer was present in cultures of long non-flagellated filaments and short non-flagellated cultures grown at suboptimal temperatures. The amount of flagellin present appeared to be equal in both non-flagellated and flagellated cultures. When cells were grown as long non-flagellated filaments and switched to growth conditions inducing short, flagellated forms, flagella were first observed at 2.5 h after this switch.Portions of this work were previously presented at the 91st General Meeting of the American Society for Microbiology, May 5–9 1991, Dallas, Texas (abstract I-81) and at the 41st Annual Meeting of the Canadian Society of Microbiologists, June 3–6 1991, London, Ontario (Abstract MP-1)     

Glutamate Metabolism in a Glutamate-producing Bacterium,Brevibacterium flavum

Brevibacterium flavum No. 2247 was found to grow with l-glutamate as the sole carbon and nitrogen source on an agar-plate medium when high concentrations of l-glutamate, FeSO₄ and biotin were added to the medium. It grew on l-glutamate in liquid medium only when yeast extract or high concentrations of FeSO₄ and glucose or organic acids of the tricarboxylic acid cycle were added to the medium. The growth on l-glutamate in liquid medium was also stimulated by high concentrations of l-glutamate, biotin and MgSO₄, and inhibited by a high concentration of (NH₄)₂SO₄.

Aspartate aminotransferase (TA)- and α-ketoglutarate dehydrogenase (KD)-defective mutants did not grow on l-glutamate, and glutamate-utilizing revertants derived from these mutants recovered TA and KD activity, respectively, whereas glutamate dehydrogenase (GD)-defective mutants grew on l-glutamate. Washed cells of strain No. 2247 grown on glutamate decomposed the amino acid, whereas those grown on glucose did not. The degradation was observed only under aerobic conditions. The former cells showed higher KD, succinate dehydrogenase and fumarase activities than the latter cells. Of 75 mutants which did not grow on glutamate but grew on succinate, three strains lacked KD but showed the same glutamate productivity as the parent strain. Four other strains with normal KD levels showed higher glutamate productivity than the parent.     

Primary Root Growth and the Pattern of Root Apical Meristem Organization are Coupled

Root apical meristems (RAMs) in dicotyledonous plants have two organizational schemes; closed (with highly organized tiers) and open (tiers lacking or disorganized). These schemes are commonly believed to remain unchanged during the growth of the root axis. Individual roots are commonly thought to have indeterminate growth. We challenge these two generalizations through the study of five species with closed apical organization: Clarkia unguiculata L., Oxalis corniculata L., Dianthus caryophyllus L., Blumenbachia hieronymi Urb., and Salvia farinaceae Benth. cv. Strata. These roots have phased growth patterns where early growth is followed by deceleration, after which the initial cells stop dividing, elongation ceases, and the root reaches its determinate length. At or before reaching determinacy, the root apical meristem stops maintaining its closed organization and becomes less organized. These observations will be placed in context with observations from the literature to suggest two new generalizations, namely, that apical organization does change over the growth phases of roots, and that roots are determinate.     

Structure of occluding junctions in ileal epithelial cells of suckling rats

Summary Two kinds of occluding junctions are found between ileal epithelial cells of suckling rats: apical zonulae occludentes (ZO) and fasciae occludentes (FO) which are associated with the lateral plasma membranes of many epithelial cells. In unfixed preparations, glycerol treatment induces the further proliferation of extensive fasciae occludentes. Both kinds of junction have identical structural elements when visualized in freeze fracture replicas, although the arrangement of these elements differs. Zonulae occludentes consist of networks of branching and anastomosing linear ridges or rows of 10 nm particles with 20–30 nm spaces between the rows which form narrow belt-like structures around the apical region of adjacent cells. Fasciae occludentes, on the other hand, consist of similar linear ridges or rows of particles but the junction strands are often discontinuous, open ended and only occasionally intersect with each other. Several different fracture planes through the plasma membrane in the region of the occluding junctions have been observed and these provide further evidence that two components, one from each membrane, fused at the level of the extracellular space, form the junction sealing element. Furthermore, we present evidence which indicates a staggered rather than an in-register arrangement of these two components.This study was supported in part by National Institutes of Health Program Project No. NS10299 and National Institutes of Health Sciences Advancement Award No. RR06148 (J.D.R.) and by the Cancer Research Campaign (S.K.) and Medical Research Council (A.R.L.)     

A covert contaminant of cultured plant cells: elimination of a Hyphomicrobium sp. from cultures of Datura innoxia (Mill.)

We have discovered a bacterial contaminant in some cell cultures of Datura innoxia (Mill.). The bacterium was tentatively identified as a species of Hyphomicrobium on the basis of its morphology and life cycle, and was isolated and grown in pure culture on a defined medium. The contaminant was not macroscopically observable in plant cell cultures. It caused neither a reduction of plant cell growth nor a noticeable increase in culture turbidity. Furthermore, it was not readily detectable by many standard assays for culture contamination: it would not grow alone in plant culture medium or yeast extract potato dextrose medium, and grew only very slowly on nutrient agar or beef-peptone medium. Repeated treatments with a combination of streptomycin (100 g/ml) and carbenicillin (100 g/ml) eliminated the contaminant from D. innoxia cell cultures without harming the plant cells.     

A comparison of the lytic action ofCytophaga johnsonii on a eubacterium and a yeast

The non-fruiting myxobacteriumC. johnsonii will not attack living cells ofAerobacter aerogenes orSaccharomyces cerevisiae. Autoclaved cells of both organisms are however lysed but in different ways. WithA. aerogenes the cell contents are dissolved leaving an outer membrane which is not further attacked. With the yeast the wall is lysed and a structure resembling a spheroplast remains. Glucanase is produced by the myxobacterium when grown on autoclaved whole yeast. No glucanase is produced when the organism is grown on autoclavedA. aerogenes.     

High intracellular expression of giant catfish growth hormone under the control of PGK promoter in Saccharomyces cerevisiae

Different yeast plasmid systems containing different promoters such as ADH1, PGK, GAPDH and GAL1, and different selectable markers, such as URA3, TRP1 and leu2-d were compared to obtain the yeast expression system that provides high intracellular expression of giant catfish growth hormone (gcGH). The highest level of gcGH expression was observed in a recombinant yeast under the control of PGK promoter (17.1 mg/l or 1.4 g/0.1 OD). The amount of gcGH was increased six-fold (102.5 mg/l) when cells were grown in a rich medium (YEPD) with the inoculum and medium ratio of 1:1, although the amount of gcGH expression per cell density did not increase (1.0 g/0.1 OD). This indicated that the increased yield of gcGH in rich medium was due to the increased cell density. The aim of the study was to produce high level gcGH in the cells of S. cerevisiae in order to use the yeast cells as potential feed additives to promote growth in giant catfish.     

Expression of a gene (Ids2) induced by Fe deficiency treatment in the roots of Hordeum vulgare L.

A ZAPII-cDNA library constructed from poly(A)⁺-RNA isolated from Fe-deficient barley roots was used for differential screening of barley roots grown in the presence and absence of Fe. Among seven clones that hybridised specifically to the probe for Fe deficiency, one clone (Ids2) was sequenced. Using a part of the cDNA sequence as a probe, a genomic-DNA library was probed and a corresponding DNA clone was isolated and sequenced. The predicted amino acid sequence resembled 2-oxoglutarate-dependent dioxygenase. Ids2 had a metal regulatory element as well as Cu regulatory elements of CUP1 gene of yeast MT.     

Cell elongation and cell division in elongating lettuce hypocotyl sections

The roles of cell division and cell elongation in the growth of sections excised from hypocotyls of lettuce (Lactuca sativa L. cv. Arctic) were investigated. Elongation of sections incubated in the light is inhibited compared to dark-grown sections and this inhibition is reversed by gibberellic acid (GA₃). The elongation of both dark-grown and GA₃-treated, light-grown sections can be enhanced by 10mM KCl. Under all conditions of incubation, elongation growth is greatest in the uppermost quarter of the hypocotyl section while the basal quarter does not elongate. In darkness the two apical segments of sections marked into four equal parts grow at the same rate, while in light, growth of the apical segment exceeds that of the second segment. Cell division in cortical or epidermal cells, as measured by mitotic index or cell number, is not affected by illumination conditions nor by GA₃ or KCl treatments. Although -irradiation and FUDR pretreatment eliminate or cause a marked reduction in cell division in the excised hypocotyl, sections from seeds irradiated with -rays or incubated in 5-fluorodeoxyuridine elongate in response to GA₃ and KCl treatment as do sections from non-pretreated controls. Therefore, since neither GA₃ nor darkness affect celldivision activity and since treatments which eliminate or significantly reduce cell division do not affect growth, we conclude that the effect of GA₃ and darkness in this material is to increase cell elongation.Abbreviations FUDR 5-fluorodeoxyuridine - GA(s) gibberellin(s) - GA₃ gibberellic acid     

Effect of endocytosis upon acid phosphatase activity ofTetrahymena pyriformis

Summary Increased endocytosis inTetrahymena pyriformis, produced by presenting starved cells with either peptone-yeast extract medium or killed yeast cell suspension, results in increased cellular acid phosphatase activity.Tetrahymena, grown in peptone-yeast extract medium, showed increased acid phosphatase activity after phagocytosis of yeast cells. This increase was not apparent until about one hour after presentation and was maximal at about 2.5 hours.Tetrahymena, grown on yeast suspension, showed little increase in acid phosphatase activity on presentation with peptone-yeast extract medium. These results may indicate that endocytosis, of either particles or solutes, produces an adaptive increase in acid phosphatase activity (presumably lysosomal in nature) which is related to feeding.Histochemical examination failed to localise the increase in acid phosphatase activity cellularly, but small particles, of about 1 diameter, which showed acid phosphatase activity and were presumably lysosomes were noted. Closely orientated yeast cells showed varying intensities of lead deposition, from absence to intense staining. This suggests that newly ingested yeast cells may be ingested initially in a single phagosome and that thereafter one or more lysosomes may fuse with them.     

Degradation of pyrimidine bases in Clostridium sticklandii

Resting cells of Clostridium sticklandii took up thymine or uracil, when grown in a medium containing 40 mM serine and 20 mM thymine or uracil. The uptake was much lower, when the cells had been grown in a complex medium. Cell-free extracts from cells grown in the complex medium reduced the two bases to the dihydro compounds and decomposed dihydrothymine to -ureidoisobutyrate, as indicated by thin-layer chromatography. Uptake and degradation were stimulated by both NADH and NADPH. Further breakdown did not occur, as ¹⁴CO₂ was not evolved from C-2-labelled thymine or uracil. The rates of pyrimidine uptake and breakdown of C. sticklandii were lower than those reported for C. sporogenes (Hilton et al., 1975).     

Environmental and developmental regulation of carotenogenesis in the dimorphic fungus Mucor rouxii

Aerobic mycelium of wild-type Mucor rouxii accumulated about ten times higher amounts of the carotenoid pigment -carotene when grown continuously in the presence of light than the corresponding cultures grown in the dark. Carotenoid accumulation was dependent on light intensity, with the threshold located at about 10^-2 W.m^-2. Photocarotenogenesis in complex medium was more efficient with glucose as a carbon source. Carotenoid synthesis by M. rouxii mycelium was unaffected by both retinol acetate and retinal, which are stimulators of carotenogenesis in other zygomycetes. Carotenogenesis was significant in aerobic mycelium but was almost undetectable in anaerobic mycelium as well as in aerobic or anaerobic yeast cells. This suggested an involvement of oxygen in carotenoid synthesis by M. rouxii and the existence of developmental regulation of the expression or operation of the pathway.     

The interaction of phagocytes and the large-sized parasite Cryptococcus neoformans: Cytochemical and ultrastructural study

Summary The yeast Cryptococcus neoformans may develop under certain conditions a large polysaccharide capsule 50–100 M in diameter and therefore cannot be phagocytosed by either polymorphonuclear cells (PMN's) or mononuclear phagocytes (MN's). The cellular defense mechanism — in various animals — against the yeast is composed by formation of ringlike structure of PMN's or MN's cells which surround the C. neoformans. Ring structures develop either in vivo or in vitro in tissue culture; destruction of the yeast occurs within 36–72 hours.Several hydrolases, such as acid phosphatase, -glucuronidase and non-specific esterase were found to be released from the phagocytic cells into the enclosed yeast. Considerable reduction of NBT used as a marker for oxidative activity was observed in MN rings at contact regions of the MN cells and the yeast. Electron microscopic studies indicate that the phagocytic cells in the ring structure have many pseudopodes penetrating into the polysaccharide capsule of the yeast. Disintegration of the capsule was observed as well as phagocytosis of its material. A possible analogy between normal phagocytosis of small-sized bodies and the ring structure obtained when large bodies are involved is discussed.     

Superoxide dismutase is involved in high tolerance to copper in the deep-sea yeast, Cryptococcus sp. N6

The activity and expression of superoxide dismutase (SOD) was analyzed in a copper-tolerant yeast, Cryptococcus sp. N6. Using cell extracts, two distinct bands exhibiting SOD activity appeared on native PAGE: one band, with higher mobility, appeared when the cells were grown without CuSO₄, and the other band appeared when the cells were grown with 10 mM CuSO₄. Cells grown with 3 mM CuSO₄ produced both SOD isoforms. Western blot analysis, using a monoclonal antibody against human SOD-1, showed that SOD protein was expressed in the absence of CuSO₄ and that the expression level increased when the cells were grown with 3 or 10 mM CuSO₄. The molecular weight of SOD from strain N6 was approx. 18 kDa. Treatment of the cells with the protein synthesis inhibitor, cycloheximide at 0.5 g ml^–1, did not affect cell growth in the absence of CuSO₄ but significantly inhibited growth in the presence of 10 mM CuSO₄ and inhibited expression of SOD protein. This suggests that SOD may play a role in cell growth in the presence of high concentrations of CuSO₄.     

A dioxygenase gene (Ids2) expressed under iron deficiency conditions in the roots of Hordeum vulgare

A zapII cDNA library was constructed from mRNA isolated from Fe-deficient barley roots and screened with cDNA probes made from mRNA of Fe-deficient and Fe-sufficient (control) barley roots. Seven clones were selected. Among them a clone having the putative full-length mRNA of dioxygenase as judged by northern hybridization was selected and named Ids2 (iron deficiency-specific clone 2). Using a cDNA fragment as probe, two clones from the genomic library (EMBL-III) were isolated and one was sequenced. The predicted amino acid sequence of Ids2 resembled that of 2-oxoglutarate-dependent dioxygenase. Ids2 is expressed in the Fe-deficient barley roots but is not in the leaves. The expression is repressed by the availability of Fe. Ids2 was also strongly expressed under Mn deficiency and weakly under Zn deficiency or excess NaCl (0.5%). The upstream 5-flanking region of Ids2 has a root-specific cis element of the CaMV 35S promoter and a nodule-specific element of leghemoglobin, a metal regulatory element (MRE) and several Cu regulatory elements (UAS) of yeast metallothionein (CUP1).     

Effects of concentration and treatment duration upon dwarf pea response to gibberellic acid root treatments in solution culture

Gibberellic acid (GA₃) root treatments stimulated internode elongation of hydroponically grown dwarf pea seedlings (Pisum sativum L.,cv. Little Marvel) When the GA₃ concentration in the solution was at least 2.9 M.Both GA₃ concentration and the duration of the root-treatment period significantly affected internode elongation. This is attributed to a limited availability or saturation of active sites for gibberellin-induced cell elongation. The amount of GA₃ taken up through the roots in 1 day from a 29 M GA₃ solution apparently equaled or exceeded the amount which could be metabolized during the first four days after treatment, although higher concenrations and longer treatment periods produced a more prolonged response, conceivably due to 1) initial saturation of gibberellin active sites, 2) storage of surplus gibberellin in the plant, and 3) subsequent utilization of the stored gibberellin. GA₃-induced stem elongation in hydroponically grown Little Marvel peas seemed to be limited initially by apparent saturation of active sites when the GA₃ concentration exceeded 29 M.     

Utilization of malate or aspartate by a yeast lacking malate enzyme

Summary Hansenula anomala, a yeast lacking malate enzyme, was able to grow in media containing malate or aspartate as sole carbon and energy sources. Both aspartate--ketoglutarate transaminase and pyruvate kinase activities changed their levels when the yeast was grown on different carbon sources. Pyruvate kinase activity was increased by fructose 1,6-diphosphate.These results indicate that in this yeast malate enzyme is not indispensable for the formation of pyruvate from malate or aspartate and that C₄ dicarboxylic acids may provide pyruvate through the combined action of phosphoenolpyruvate carboxykinase and pyruvate kinase. It is also concluded that aspartate--ketoglutarate transaminase and pyruvate kinase are under regulatory control in Hansenula anomala.     

Expression of the rice Osgrp1 promoter-Gus reporter gene is specifically associated with cell elongation/expansion and differentiation

To study the expression and regulation of a rice glycine-rich cell wall protein gene, Osgrpl, transgenic rice plants were regenerated that contain the Osgrpl promoter or its 5 deletions fused with the bacterial -glucuronidase (GUS) reporter gene. We report here a detailed histochemical analysis of the Osgrpl-Gus expression patterns in transgenic rice plants. In roots of transgenic rice plants, GUS expression was specifically located in cell elongation and differentiation regions, and no GUS expression was detectable in the apical meristem and the mature region. In shoots, GUS activity was expressed only in young leaves or in the growing basal parts of developing leaves, and little GUS activity was expressed in mature leaves or mature parts of developing leaves. In shoot apices, GUS activity was detected only in those leaf cells which were starting to expand and differentiate, and GUS expression was not detected in the apical meristem and the young meristematic leaf primordia. GUS activity was highly expressed in the young stem tissue, particularly in the developing vascular bundles and epidermis. Thus, the expression of the Osgrpl gene is closely associated with cell elongation/expansion during the post-mitotic cell differentiation process. The Osgrpl-Gus gene was also expressed in response to wounding and down-regulated by water-stress conditions in the elongation region of roots. Promoter deletion analysis indicates that both positive and negative mechanisms are involved in regulating the specific expression patterns. We propose a simple model for the developmental regulation of the Osgrpl gene expression.     

Apical abortion in calabrese is induced by periods of low temperature and results in premature differentiation of apical meristem cells

Apical abortion in calabrese (Brassica oleracea var. italica), a highly destructive disorder which occurs in overwintered transplants, has been investigated using a model system in which blindness (abortion of the apical meristem) can be reproducibly and predictably induced. An initial experiment examined the susceptibility of 12 cultivars to apical abortion when grown throughout a winter period under commercial conditions. Three of those varieties showed very high levels of blindness (100%). Subsequently, plants of the susceptible cultivar PETO 7204 were subjected to an inductive period of low light intensity (30 mol m^-2 s^-1) and low temperature (4 C). Apical meristematic cells of all plants ceased mitotic activity within 3 d of being transferred to a regime comprising higher light intensity (100 mol m^-2 s^-1) and temperature (15 C). Using this system the structures of normal apices were compared with those which became blind. Blindness was characterized by a cessation of leaf primordium production by the vegetative apex, the last formed primordium growing on in some cases to form a mature normal leaf, or in others, a deformed structure known as a whip-tail. The inactive apical bud became embedded in the tissues of this last-formed structure. The cells of the inactivated apical bud remained alive, but lost their meristematic capability, becoming enlarged, highly vacuolated parenchyma cells with amyloplasts.Keywords: Apical abortion, apical meristem, blindness, calabrese.