Plants show varied cellular responses to salinity that are partly associated with maintaining low cytosolic Na+ levels and a high K+/Na+ ratio. Plant metabolites change with elevated Na+, some changes are likely to help restore osmotic balance while others protect Na+-sensitive proteins. Metabolic responses to salt stress are described for two barley (Hordeum vulgare L.) cultivars, Sahara and Clipper, which differed in salinity tolerance under the experimental conditions used. After 3 weeks of salt treatment, Clipper ceased growing whereas Sahara resumed growth similar to the control plants. Compared with Clipper, Sahara had significantly higher leaf Na+ levels and less leaf necrosis, suggesting they are more tolerant to accumulated Na+. Metabolite changes in response to the salt treatment also differed between the two cultivars. Clipper plants had elevated levels of amino acids, including proline and GABA, and the polyamine putrescine, consistent with earlier suggestions that such accumulation may be correlated with slower growth and/or leaf necrosis rather than being an adaptive response to salinity. It is suggested that these metabolites may be an indicator of general cellular damage in plants. By contrast, in the more tolerant Sahara plants, the levels of the hexose phosphates, TCA cycle intermediates, and metabolites involved in cellular protection increased in response to salt. These solutes remain unchanged in the more sensitive Clipper plants. It is proposed that these responses in the more tolerant Sahara are involved in cellular protection in the leaves and are involved in the tolerance of Sahara leaves to high Na+.

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The plasma membrane of Mycobacterium sp. is the site of synthesis of several distinct classes of lipids that are either retained in the membrane or exported to the overlying cell envelope. Here, we provide evidence that enzymes involved in the biosynthesis of two major lipid classes, the phosphatidylinositol mannosides (PIMs) and aminophospholipids, are compartmentalized within the plasma membrane. Enzymes involved in the synthesis of early PIM intermediates were localized to a membrane subdomain termed PM f , that was clearly resolved from the cell wall by isopyknic density centrifugation and amplified in rapidly dividing Mycobacterium smegmatis. In contrast, the major pool of apolar PIMs and enzymes involved in polar PIM biosynthesis were localized to a denser fraction that contained both plasma membrane and cell wall markers (PM-CW). Based on the resistance of the PIMs to solvent extraction in live but not lysed cells, we propose that polar PIM biosynthesis occurs in the plasma membrane rather than the cell wall component of the PM-CW. Enzymes involved in phosphatidylethanolamine biosynthesis also displayed a highly polarized distribution between the PM f and PM-CW fractions. The PM f was greatly reduced in non-dividing cells, concomitant with a reduction in the synthesis and steady-state levels of PIMs and amino-phospholipids and the redistribution of PM f marker enzymes to non-PM-CW fractions. The formation of the PM f and recruitment of enzymes to this domain may thus play a role in regulating growth-specific changes in the biosynthesis of membrane and cell wall lipids.

Boron (B) is an essential micronutrient that affects plant growth at either deficient or toxic concentrations in soil. The aim of this work was to investigate the adaptation of barley (Hordeum vulgare) plants to toxic B levels and to increase our understanding of B toxicity tolerance mechanisms. We used a metabolomics approach to compare metabolite profiles in root and leaf tissues of an intolerant, commercial cultivar (cv Clipper) and a B-tolerant Algerian landrace (cv Sahara). After exposure to elevated B (200 and 1,000 mM), the number and amplitude of metabolite changes in roots was greater in Clipper than in Sahara. In contrast, leaf metabolites of both cultivars only responded following 1,000 mM treatment, at which B toxicity symptoms (necrosis) were visible. In addition, metabolite levels were dramatically altered in the tips of leaves of the sensitive cultivar Clipper after growth in 1,000 mM B compared to those of Sahara. This correlates with a gradual accumulation of B from leaf base to tip in B-intolerant cultivars. Overall, there were always greater differences between tissue types (roots and leaves) than between the two cultivars. This work has provided insights into metabolic differences of two genetically distinct barley cultivars and information about how they respond metabolically to increasing B levels.Boron (B) is an essential micronutrient for vascular plants. However, when B is present at high concentrations in the soil or ground water, plant growth and reproduction can be affected by B toxicity. B toxicity has been recognized as an important problem limiting crop production in the low rainfall and on highly alkaline and saline soils in regions of Australia, West Asia, and North Africa. Because soil amelioration is impractical, the development of B-tolerant cultivars is a rational solution to the problem.B freely diffuses into the roots as boric acid [B(OH) 3 ; pK a 5 9.25] and accumulates in the cytoplasm as the borate anion [B(OH) 4 2 ] due to pH-dependent interconversion. An inability to exclude B from the roots results in high B concentrations in the tissue. B phytotoxicity manifests itself in a broad range of physiological effects, including decreased shoot and root growth, root cell division and RNA content, reduced leaf chlorophyll, lower photosynthetic rates and stomatal conductance, and reduced levels of lignin and suberin (for review, see Nable et al., 1997). Leaf symptoms of toxicity in barley (Hordeum vulgare) are characterized by interveinal chlorotic and/or necrotic patches, generally at the margins and tips of older leaves. This reflects the accumulation of B at the end of the transpiration stream (Nable et al., 1997). Following long-term exposure to high B concentrations in the soil, overall vegetative plant growth is retarded and this leads to either a reduction in or a complete lack of seed set.B is also an essential nutrient, although its role in plant growth, development, and metabolism remains to be clarified. Originally, B was thought to be essentially immobile in the ...

All mycobacterial species, including pathogenic Mycobacterium tuberculosis, synthesize an abundant class of phosphatidylinositol mannosides (PIMs) that are essential for normal growth and viability. These glycolipids are important cell-wall and/or plasma-membrane components in their own right and can also be hyperglycosylated to form other wall components, such as lipomannan and lipoarabinomannan. We have investigated the steps involved in the biosynthesis of the major PIM species in a new M. smegmatis cell-free system. A number of apolar and polar PIM intermediates were labelled when this system was continuously labelled or pulse-chase-labelled with GDP-[3H]Man, and the glycan head groups and the acylation states of these species were determined by chemical and enzymic treatments and octyl-Sepharose chromatography respectively. These analyses showed that (1) the major apolar PIM species, acyl-PIM2, can be synthesized by at least two pathways that differ in the timing of the first acylation step, (2) early PIM intermediates containing a single mannose residue can be modified with two fatty acid residues, (3) formation of polar PIM species from acyl-PIM2 is amphomycin-sensitive, indicating that polyprenol phosphate-Man, rather than GDP-Man, is the donor for these reactions, (4) modification of acylated PIM4 with alpha1-2- or alpha1-6-linked mannose residues is probably the branch point in the biosyntheses of polar PIM and lipoarabinomannan respectively and (5) GDP strongly inhibits the synthesis of early PIM intermediates and increases the turnover of polyprenol phosphate-Man. These findings are incorporated into a revised pathway for mycobacterial PIM biosynthesis.

RNA encoding the rat serotonin 5-HT

All species of Mycobacteria synthesize distinctive cell walls that are rich in phosphatidylinositol mannosides (PIMs), lipomannan (LM), and lipoarabinomannan (LAM). PIM glycolipids, having 2-4 mannose residues, can either be channeled into polar PIM species (with 6 Man residues) or hypermannosylated to form LM and LAM. In this study, we have identified a Mycobacterium smegmatis gene, termed lpqW, that is required for the conversion of PIMs to LAM and is highly conserved in all mycobacteria. A transposon mutant, Myco481, containing an insertion near the 3 end of lpqW exhibited altered colony morphology on complex agar medium. This mutant was unstable and was consistently overgrown by a second mutant, represented by Myco481.1, that had normal growth and colony characteristics. Biochemical analysis and metabolic labeling studies showed that Myco481 synthesized the complete spectrum of apolar and polar PIMs but was unable to make LAM. LAM biosynthesis was restored to near wild type levels in Myco481.1. However, this mutant was unable to synthesize the major polar PIM (AcPIM6) and accumulated a smaller intermediate, AcPIM4. Targeted disruption of the lpqW gene and complementation of the initial Myco481 mutant with the wild type gene confirmed that the phenotype of this mutant was due to loss of LpqW. These studies suggest that LpqW has a role in regulating the flux of early PIM intermediates into polar PIM or LAM biosynthesis. They also suggest that AcPIM4 is the likely branch point intermediate in polar PIM and LAM biosynthesis.Members of the genus Mycobacterium cause important diseases in humans, including tuberculosis and leprosy. Mycobacterium tuberculosis is thought to infect nearly one-third of the world population and to cause two to three million deaths each year (1). This threat to global health is growing as drug-resistant strains emerge and coinfections with human immunodeficiency virus increase the number of individuals with active tuberculosis. All species of mycobacteria synthesize a highly distinctive cell wall that contributes to the ability of pathogenic mycobacteria to survive within the endosomal network of human macrophages and to their innate resistance to many antibiotics. The mycobacterial cell wall has a multilaminate structure, comprising an asymmetric outer membrane and an inner layer of arabinogalactan polysaccharide and peptidoglycan (2, 3). The asymmetric outer membrane has an inner leaflet of tightly packed, long chain (C70 -C90) mycolic acids and an outer leaflet of free (glyco)lipids. This asymmetric outer membrane is responsible for the low permeability properties of the cell wall and also contains lipids that play key roles in the pathogenesis of these organisms.Although the (glyco)lipid composition of the mycobacterial cell wall can vary among mycobacterial species, all species synthesize an abundant class of phosphatidylinositol mannosides (PIMs) 7 and the hypermannosylated PIMs, lipomannan (LM) and lipoarabinomannan (LAM) (4, 5). The PIMs, LM and LAM, may be located in the plasm...

Glycopeptidolipids (GPLs) are major components of the cell walls of several species of mycobacteria. We have isolated a transposon mutant of Mycobacterium smegmatis that is unable to synthesize mature GPLs and that displays a rough colony morphology. The disrupted gene, mtf1, shares a high degree of homology with several S-adenosylmethionine-dependent methyltransferases. The enzyme encoded by mtf1 is required for the methylation of a single rhamnose residue that forms part of the conserved GPL core structure. This conclusion is supported by the finding that (a) the mutant synthesized only GPLs with undermethylated (either mono-or nonmethylated instead of di-or trimethylated) rhamnose residues; (b) complementation of the mutant with a wild-type copy of mtf1 restored high levels of synthesis of GPLs containing di-and trimethylated rhamnose; and (c) S-adenosylmethionine-dependent methylation of rhamnosylated GPLs could be detected in cell lysates of wild-type cells and mtf1-complemented mutant cells, but not in mutant cells lacking intact mtf1. Structural analysis of wild-type and mutant GPLs suggests that disruption of mtf1 specifically inhibits addition of O-methyl groups to the 3 (or 2)-position of the rhamnose. In the absence of 3-O-methylation, further methylation of GPL rhamnose is apparently inhibited, and overall GPL synthesis is down-regulated by 90%.Several species of mycobacteria cause important human diseases. For example, Mycobacterium tuberculosis is the causative agent of tuberculosis, the leading cause of death from a single bacterial infection, whereas species of the Mycobacterium avium complex cause intractable infections in immunocompromised individuals (1). Many features of mycobacteria, including their ability to proliferate within phagolysosomes of host macrophages and their general resistance to a wide range of antibiotics, have been attributed to the fact that all these organisms synthesize distinctive lipid-rich cell walls (1-3). In addition to forming a highly effective permeability barrier, specific components in this wall have been shown to contribute to pathogenesis and/or to mediate specific host-bacterial interactions (4). The mycobacterial cell wall is composed of a core peptidoglycan-arabinogalactan layer surrounded by an outer lipid bilayer. The inner leaflet of the lipid bilayer is composed of mycolic acids, whereas several distinct classes of glycolipids and phospholipids form the outer leaflet (2, 3). The outer layer glycolipids are thought to contribute to the distinct surface properties of the different mycobacterial species and are also important surface antigens. The predominant outer layer glycolipids in members of the M. avium complex are glycopeptidolipids (GPLs), 1 which characteristically contain a tripeptideamino alcohol core that is modified with an amide-linked fatty acid, a 6-dTal residue, and a variably O-methylated Rha residue ( Fig. 1) (5-7). GPLs having this core structure are termed non-serovar-specific GPLs (nsGPLs) and are found in most isolates of the M. avi...