Structural elucidation of the predominant motifs of the major cell wall arabinogalactan antigens from the borderline species Tsukamurella paurometabolum and Mycobacterium fallax

Tsukamurella paurometabolum and Mycobacterium fallax are members of the suprageneric actinomycete group Corynebacterineae that possesses a cell wall skeleton composed of a peptidoglycan to which an arabinogalactan is covalently attached. This polysaccharide is further modified by esterification with C60-C80 mycolic acid residues in mycobacteria and T. paurometabolum. However, M. fallax and T. paurometabolum produce polyenoic (up to six double bonds) mycolic acids whereas the most common type of mycobacterial mycolates, called alpha-mycolates, are mono- and di-enoic or -cyclopropanated mycolic acids. To determine whether this difference also applied to the structures of cell wall arabinogalactans, competitive inhibition experiments using antibodies raised against the cell wall from Mycobacterium bovis and the arabinogalactans from T. paurometabolum and M. fallax were performed. They demonstrated the structural identity between the polysaccharide of M. fallax and those of mycobacteria and showed a strong similarity between the latter polysaccharides and that of T. paurometabolum. Structural analyses of the per-O-alkylated alditol fragments derived from the polysaccharides by gas chromatography-mass spectrometry (GC-MS) and 13C nuclear magnetic resonance (NMR) spectroscopy of the intact solubilized polysaccharides demonstrated that the polysaccharides from the two species analyzed contained all the major structural features previously characterized in mycobacterial arabinogalactans. These include (1) the homogalactan of alterning 5-linked galactofuranosyl (Galf) and 6-linked Galf residues, (2) a linear 5-linked arabino furanosyl (Araf), (3) a beta-Araf-(1-->2)-alpha-Araf disaccharide branched on both position 3 and position 5 of an alpha-Araf unit, and (4) a 5-linked-alpha-Araf unit branched on both position 3 and position 5 of an alpha-Araf residue. The polysaccharide from T. paurometabolum possesses additional structural domains composed of a terminal (t) Araf directly linked to either a 5-linked-alpha-Araf or to both position 3 and position 5 of a 3,5-linked alpha-Araf unit. Both the remarkable similarity of arabinogalactans from Corynebacterineae and their genus- and/or species-specificities are reflected in their 13C NMR spectra that may be used as a valuable help in the identification of members of the actinomycete group.     

Cell wall composition of chlorococcal algae

The cell walls of representatives of the genera Chlorella, Monoraphidium, Ankistrodesmus and Scenedesmus contained 24–74% neutral sugars, 1–24% uronic acids, 2–16% protein and 0–15% glucosamine. Two types of cell walls could be discerned containing as main sugars either rhamnose and galactose or mannose and glucose with a lack of galactose.     

Topology and mutational analysis of the single Emb arabinofuranosyltransferase of Corynebacterium glutamicum as a model of Emb proteins of Mycobacterium tuberculosis

The cell wall mycolyl-arabinogalactan (AG)--peptidoglycan complex is essential in mycobacterial species, such as Mycobacterium tuberculosis, and is the target of several antitubercular drugs. For instance, ethambutol (EMB) targets AG biosynthesis through inhibition of the arabinofuranosyltransferases Mt-EmbA and Mt-EmbB, as well as the single Emb from Corynebacterium glutamicum. Here, we present for the first time an experimental analysis of the membrane topology of Emb. The domain organization clearly positions highly conserved loop regions, like the recognized glycosyltransferase C motif and the hydrophilic C-terminus towards the periplasmic side of the cell. Moreover, the assignment and orientation of hydrophobic segments identified a loop region, which might dip into the membrane and could possibly line a transportation channel for the emerging substrate. Site-directed mutations introduced into plasmid-encoded Cg-emb were analyzed in a C. glutamicumDeltaemb strain for their AG glycosyl composition and linkage analysis. Mutations analyzed did not perturb galactan synthesis; however, D297A produced a dramatically reduced arabinan content and prevented growth, indicating an inactive Emb. A second D298A mutation also drastically reduced arabinan content; however, growth of the corresponding mutant was not altered, indicating a certain tolerance of this mutation in terms of Emb function. A W659L-P667A-Q674E triple mutation in the chain length regulation motif (Pro-motif) resulted in a reduced arabinose deposition in AG but retained all arabinofuranosyl linkages. Taken together, the data clearly define important residues of Emb involved in arabinan domain formation and, for the first time, shed new light on the topology of this important enzyme.     

Cell envelope stress response in Gram-positive bacteria

The bacterial cell envelope is the first and major line of defence against threats from the environment. It is an essential and yet vulnerable structure that gives the cell its shape and counteracts the high internal osmotic pressure. It also provides an important sensory interface and molecular sieve, mediating both information flow and the controlled transport of solutes. The cell envelope is also the target for numerous antibiotics. Therefore, the monitoring and maintenance of cell envelope integrity in the presence of envelope perturbating agents and conditions is crucial for survival. The underlying signal transduction is mediated by two regulatory principles, two-component systems and extracytoplasmic function sigma factors, in both the Firmicutes (low-GC) and Actinobacteria (high-GC) branches of Gram-positive bacteria. This study presents a comprehensive overview of cell envelope stress-sensing regulatory systems. This knowledge will then be applied for in-depth comparative genomics analyses to emphasize the distribution and conservation of cell envelope stress-sensing systems. Finally, the cell envelope stress response will be placed in the context of the overall cellular physiology, demonstrating that its regulatory systems are linked not only to other stress responses but also to the overall homeostasis and lifestyle of Gram-positive bacteria.     

Cell wall composition of the carrageenophyte Kappaphycus alvarezii (Gigartinales, Rhodophyta) partitioned by wet sieving

The cortical and medullary cells of Kappaphycus alvarezii fractions were screened by wet sieving after aqueous extraction of carrageenans. The cell populations obtained showed a clear partition between these two cell types. The main monosaccharide in hydro-insoluble cell walls was cellulosic glucose (70% dry weight), the crystallinity of which was shown torange from 20% in the cortical cells to 45% in the large medullary cells (over 250 μm diameter). Minor monosaccharides in the insoluble fraction were galactose, 3,6-anhydrogalactose (indicating presence of residual carrageenans), mannose and xylose. However, the major part of the remaining galactose probably originated from another galactoglycan strongly linked to insoluble polymers in the large medullarycell walls. The mannose concentration was maximum in the cortical cells and decreased with increasing size of the medullary cells. Thus, besides cellulose, two other types of polysaccharides were detected in insolublecell walls, mannoglycans and galactoglycans in cortical and medullary cellwalls, respectively. This revised version was published online in August 2006 with corrections to the Cover Date.     

Disruption of arabinogalactan proteins disorganizes cortical microtubules in the root of Arabidopsis thaliana

The cortical array of microtubules inside the cell and arabinogalactan proteins on the external surface of the cell are each implicated in plant morphogenesis. To determine whether the cortical array is influenced by arabinogalactan proteins, we first treated Arabidopsis roots with a Yariv reagent that binds arabinogalactan proteins. Cortical microtubules were markedly disorganized by 1 microM beta-D-glucosyl (active) Yariv but not by up to 10 microM beta-D-mannosyl (inactive) Yariv. This was observed for 24-h treatments in wild-type roots, fixed and stained with anti-tubulin antibodies, as well as in living roots expressing a green fluorescent protein (GFP) reporter for microtubules. Using the reporter line, microtubule disorganization was evident within 10 min of treatment with 5 microM active Yariv and extensive by 30 min. Active Yariv (5 microM) disorganized cortical microtubules after gadolinium pre-treatment, suggesting that this effect is independent of calcium influx across the plasma membrane. Similar effects on cortical microtubules, over a similar time scale, were induced by two anti-arabinogalactan-protein antibodies (JIM13 and JIM14) but not by antibodies recognizing pectin or xyloglucan epitopes. Active Yariv, JIM13, and JIM14 caused arabinogalactan proteins to aggregate rapidly, as assessed either in fixed wild-type roots or in the living cells of a line expressing a plasma membrane-anchored arabinogalactan protein from tomato fused to GFP. Finally, electron microscopy of roots prepared by high-pressure freezing showed that treatment with 5 microM active Yariv for 2 h significantly increased the distance between cortical microtubules and the plasma membrane. These findings demonstrate that cell surface arabinogalactan proteins influence the organization of cortical microtubules.     

O-methylation of chlorinated phenols in the genus Rhodococcus

The ability to O-methylate chlorinated phenols and phenol derivatives in the genus Rhodococcus was studied. Several species and strains O-methylated chlorophenols to the corresponding anisoles, namely R. equi, R. erythropolis, R. rhodochrous, and Rhodococcus sp. strains P1 and An 117. The ability for a strain to O-methylate chlorophenols did not require that it had been isolated from an environment containing a chlorinated aromatic compound. O-methylation activity was stimulated by the presence of carbohydrate. All strains preferentially O-methylated a substrate with the hydroxyl group flanked by two chlorine substitunts.     

Loss of a Functionally and Structurally Distinct ld-Transpeptidase,LdtMt5, Compromises Cell Wall Integrity in Mycobacterium tuberculosis

The final step of peptidoglycan (PG) biosynthesis in bacteria involves cross-linking of peptide side chains. This step in Mycobacterium tuberculosis is catalyzed by ld- and dd-transpeptidases that generate 3→3 and 4→3 transpeptide linkages, respectively. M. tuberculosis PG is predominantly 3→3 cross-linked, and Ldt_Mt2 is the dominant ld-transpeptidase. There are four additional sequence paralogs of Ldt_Mt2 encoded by the genome of this pathogen, and the reason for this apparent redundancy is unknown. Here, we studied one of the paralogs, Ldt_Mt5, and found it to be structurally and functionally distinct. The structures of apo-Ldt_Mt5 and its meropenem adduct presented here demonstrate that, despite overall architectural similarity to Ldt_Mt2, the Ldt_Mt5 active site has marked differences. The presence of a structurally divergent catalytic site and a proline-rich C-terminal subdomain suggest that this protein may have a distinct role in PG metabolism, perhaps involving other cell wall-anchored proteins. Furthermore, M. tuberculosis lacking a functional copy of Ldt_Mt5 displayed aberrant growth and was more susceptible to killing by crystal violet, osmotic shock, and select carbapenem antibiotics. Therefore, we conclude that Ldt_Mt5 is not a functionally redundant ld-transpeptidase, but rather it serves a unique and important role in maintaining the integrity of the M. tuberculosis cell wall.     

Distribution and chemical characterization of regular arrays in the cell walls of strains of the genus Lactobacillus

Abstract The presence of regular arrays (RAs) in the cell walls of strains of the genus Lactobacillus was examined by electron microscopy. The RAs were found in 6 species including L. bulgaricus, L. helveticus, L. acidophilus, L. fermentum, L. brevis and L. buchneri . The RAs were composed of a protein with an apparent M _r ranging from about 41000 to 55000, depending on the species upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The amino acid composition of the RA proteins was shown to be acidic and hydrophobic. The antigenicity of the RA protein from L. buchneri appeared to be specific but not common among the RA proteins from the other lactobacilli.     

Cell biology

A selection of World Wide Web sites relevant to papers published in this issue of Current Opinion in Plant Biology.     

Cell-wall lectins during winter wheat cold hardening

The polypeptide composition and functional activity of cell-wall lectins from roots of winter wheat (Triticum aestivum L., cv. Mironovskaya 808) seedlings during cold hardening were studied. Several phases of lectin activity changes were observed, which indicates their involvement in the development of general adaptation syndrome of the cell. After 0.5-h low-temperature treatment, marked alterations occurred in the profile of protein elution: lectins with mol wts of 78 and 42.5 kD disappeared and new ones with mol wts of 72, 69, 37, and 34.5 kD appeared. It was established that 17.5-and 69-kD lectins and most lectins eluted with glucose were arabinogalactan proteins (AGP), which permitted a supposition that these lectins were involved in the interaction between the cell wall and cytoskeleton. After 7-day-long hardening, total protein content reduced and lectins with mol wts of 69 and 37 kD disappeared, which corresponded to reduced lectin activity by the end of hardening. A transient appearance of 37-and 69-kD lectins, which are AGP, might indicate their involvement in the triggering the development of plant-cell defense responses.     

Inactivation of polyketide synthase and related genes results in the loss of complex lipids in Mycobacterium tuberculosis H37Rv

AIMS: Phthiocerol dimycocerosate (PDIM) waxes and other lipids are necessary for successful Mycobacterium tuberculosis infection, although the exact role of PDIM in host-pathogen interactions remains unclear. In this study, we investigated the contribution of tesA, drrB, pks6 and pks11 genes in complex lipid biosynthesis in M. tuberculosis. METHODS AND RESULTS: Four mutants were selected from M. tuberculosis H37Rv transposon mutant library. The transposon insertion sites were confirmed to be within the M. tuberculosis open reading frames for tesA (a probable thioesterase), drrB (predicted ABC transporter), pks11 (putative chalcone synthase) and pks6 (polyketide synthase). The first three of these transposon mutants were unable to generate PDIM and the fourth lacked novel polar lipids. CONCLUSIONS: Mycobacterium tuberculosis can be cultivated in vitro without the involvement of certain lipid synthesis genes, which may be necessary for in vivo pathogenicity. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of transposon mutants is a new functional genomic approach for the eventual definition of the mycobacterial 'lipidome'.     

Effect of growth media on cell envelope composition and nitrile hydratase stability in Rhodococcus rhodochrous strain DAP 96253

Rhodococcus is an important industrial microorganism that possesses diverse metabolic capabilities; it also has a cell envelope, composed of an outer layer of mycolic acids and glycolipids. Selected Rhodococcus species when induced are capable of transforming nitriles to the corresponding amide by the enzyme nitrile hydratase (NHase), and subsequently to the corresponding acid via an amidase. This nitrile biochemistry has generated interest in using the rhodococci as biocatalysts. It was hypothesized that altering sugars in the growth medium might impact cell envelope components and have effects on NHase. When the primary carbon source in growth media was changed from glucose to fructose, maltose, or maltodextrin, the NHase activity increased. Cells grown in the presence of maltose and maltodextrin showed the highest activities against propionitrile, 197 and 202?units/mg cdw, respectively. Stability of NHase was also affected as cells grown in the presence of maltose and maltodextrin retained more NHase activity at 55?°C (45 and 23?%, respectively) than cells grown in the presence of glucose or fructose (19 and 10?%, respectively). Supplementation of trehalose in the growth media resulted in increased NHase stability at 55?°C, as cells grown in the presence of glucose retained 40?% NHase activity as opposed to 19?% without the presence of trehalose. Changes in cell envelope components, such mycolic acids and glycolipids, were evaluated by high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC), respectively. Changing sugars and the addition of inducing components for NHase, such as cobalt and urea in growth media, resulted in changes in mycolic acid profiles. Mycolic acid content increased 5 times when cobalt and urea were added to media with glucose. Glycolipids levels were also affected by the changes in sugars and addition of inducing components. This research demonstrates that carbohydrate selection impacts NHase activity and stability. Cell envelope components such as mycolic acids are also influenced by sugars and inducers such as cobalt and urea. This is information that can be useful when implementing rhodococcal catalysts in industrial applications.     

Variations of the Surface Sculpture and Cell Wall Ultrastructure of the Zygospores in Chlamydomonas geitleri (Chlorophyta)

The superficial cell wall ornamentation in the zygospores of the alga Chlamydomonas geitleri Ettl (Chlorophyta) is formed by thickenings of the cell wall which are shaped into a network of anastomosing ribs, sometimes with local wart-like protuberances. Clearly different sculpture patterns (given by presence, arrangement and/or morphological modification of sculpture elements) were accompanied by many transient forms. Sculpture variations occurred even in clonal cultures. In the zygospore cell wall of C. geitleri, the inner, outer and middle layer can be distinguished from the morphological point of view. The relatively thin outer (sporopollenin) layer covers the whole surface of the zygospore wall. The thicker inner layer adhering to the zygospore protoplast forms, either solely or together with the middle layer (possessing a fine meshwork substructure), variously shaped thickening of the zygospore cell wall. Discussed are the ultrastructural morphology of the cell wall in Chlamydomonas zygospores, the striking similarity of the cell wall ultrastructure of zygospores in C. geitleri to the ultrastructure of the cell wall of vegetative cells in some green algae (subfamily Scotiellocystoideae), as well as the extensive morphological variability of the zygospore wall sculpture in C geitleri and its species specificity.     

柿果实采后软化过程中细胞壁组分代谢和超微结构的变化

柿果实采后果胶酯酶活性迅速上升,其活性与果实硬度的下降呈明显的负相关。多聚半乳糖醛酸酶活性增加缓慢,但其活性与果实硬度的下降无明显相关性。β-半乳糖苷酶活性迅速增加,其活性与果实硬度的下降呈明显的负相关。纤维素酶活性呈逐渐上升趋势,与果实硬度的下降也呈明显的负相关。伴随着细胞壁水解酶活性的增加,果实原果胶和纤维素含量迅速下降,而水溶性果胶含量则迅速上升。柿果刚采收时细胞壁结构完整,3d后细胞壁中胶层基本被溶解,甚至初生壁也局部发生降解。     

Differential Staining for Cellulosic and Modified Plant Cell Walls

A simple method to enhance the staining of cell wall components for fluorescence microscopy is described. In stems of Nicotiana tabacum and needles of Pinus eldarica lignin, the cuticle and unsaturated lipids are indicated by a purple-red fluorescence while pectocellulosic components fluorescc pale blue.     

植物原生质体的细胞壁再生

细胞壁作为植物细胞重要的组成部分,在决定细胞形状、维持机械支撑、吸收养分等方面发挥重要功能.因此,揭示植物细胞壁合成的调控机制具有重大的生物学意义.基于植物组织水平研究细胞壁的生物合成具有难以控制时间尺度、观察空间狭小等局限性.原生质体作为去除细胞壁的单个细胞是研究细胞壁再生的理想系统.在过去的几十年里报道了大量关于植...