Digestion of crystalline cellulose substrates by the clostridium thermocellum cellulosome: structural and morphological aspects.
The action of cellulosomes from Clostridium thermocellum on model cellulose microfibrils from Acetobacter xylinum and cellulose microcrystals from Valonia ventricosa was investigated. The biodegradation of these substrates was followed by transmission electron microscopy, Fourier-transform IR spectroscopy and X-ray diffraction analysis, as a function of the extent of degradation. The cellulosomes were very effective in catalysing the complete digestion of bacterial cellulose, but the total degradation of Valonia microcrystals was achieved more slowly. Ultrastructural observations during the digestion process suggested that the rapid degradation of bacterial cellulose was the result of a very efficient synergistic action of the various enzymic components that are attached to the scaffolding protein of the cellulosomes. The degraded Valonia sample assumed various shapes, ranging from thinned-down microcrystals to crystals where one end was pointed and the other intact. This complexity may be correlated with the multi-enzyme content of the cellulosomes and possibly to a diversity of the cellulosome composition within a given batch. Another aspect of the digestion of model celluloses by cellulosomes is the relative invariability of their crystallinity, together with their Ialpha/Ibeta composition throughout the degradation process. Comparison of the action of cellulosomes with that of fungal enzymes indicated that the degradation of cellulose crystals by cellulosomes occurred with only limited levels of processivity, in contrast with the observations reported for fungal enzymes. The findings were consistent with a mechanism whereby initial attack by a cellulosome of an individual cellulose crystal results in its 'commitment' towards complete degradation. (+info)
The respiratory system and diazotrophic activity of Acetobacter diazotrophicus PAL5.
The characteristics of the respiratory system of Acetobacter diazotrophicus PAL5 were investigated. Increasing aeration (from 0.5 to 4.0 liters of air min(-1) liter of medium(-1)) had a strong positive effect on growth and on the diazotrophic activity of cultures. Cells obtained from well-aerated and diazotrophically active cultures possessed a highly active, membrane-bound electron transport system with dehydrogenases for NADH, glucose, and acetaldehyde as the main electron donors. Ethanol, succinate, and gluconate were also oxidized but to only a minor extent. Terminal cytochrome c oxidase-type activity was poor as measured by reduced N, N,N,N'-tetramethyl-p-phenylenediamine, but quinol oxidase-type activity, as measured by 2,3,5,6-tetrachloro-1,4-benzenediol, was high. Spectral and high-pressure liquid chromatography analysis of membranes revealed the presence of cytochrome ba as a putative oxidase in cells obtained from diazotrophically active cultures. Cells were also rich in c-type cytochromes; four bands of high molecular mass (i.e., 67, 56, 52, and 45 kDa) were revealed by a peroxidase activity stain in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. KCN inhibition curves of respiratory oxidase activities were biphasic, with a highly resistant component. Treatment of membranes with 0.2% Triton X-100 solubilized c-type cytochromes and resulted in a preparation that was significantly more sensitive to cyanide. Repression of diazotrophic activity in well-aerated cultures by 40 mM (NH(4))(2)SO(4) caused a significant decrease of the respiratory activities. It is noteworthy that the levels of glucose dehydrogenase and putative oxidase ba decreased 6. 8- and 10-fold, respectively. In these cells, a bd-type cytochrome seems to be the major terminal oxidase. Thus, it would seem that glucose dehydrogenase and cytochrome ba are key components of the respiratory system of A. diazotrophicus during aerobic diazotrophy. (+info)
Acid hydrolysis of bacterial cellulose reveals different modes of synergistic action between cellobiohydrolase I and endoglucanase I.
Intact and partially acid hydrolyzed cellulose from Acetobacter xylinum were used as model substrates for cellulose hydrolysis by 1,4-beta-D-glucan-cellobiohydrolase I (CBH I) and 1,4-beta-D-endoglucanase I (EG I) from Trichoderma reesei. A high synergy between CBH I and EG I in simultaneous action was observed with intact bacterial cellulose (BC), but this synergistic effect was rapidly reduced by acid pretreatment of the cellulose. Moreover, a distinct synergistic effect was observed upon sequential endo-exo action on BC, but not on bacterial microcrystalline cellulose (BMCC). A mechanism for endo-exo synergism on crystalline cellulose is proposed where the simultaneous action of the enzymes counteract the decrease of activity caused by undesirable changes in the cellulose surface microstructure. (+info)
Identification of essential amino acids in the bacterial alpha -mannosyltransferase aceA.
The alpha-mannosyltransferase AceA from Acetobacter xylinum belongs to the CaZY family 4 of retaining glycosyltransferases. We have identified a series of either highly conserved or invariant residues that are found in all family 4 enzymes as well as other retaining glycosyltransferases. These residues included Glu-287 and Glu-295, which comprise an EX(7)E motif and have been proposed to be involved in catalysis. Alanine replacements of each conserved residue were constructed by site-directed mutagenesis. The mannosyltransferase activity of each mutant was examined by both an in vitro transferase assay using recombinant mutant AceA expressed in Escherichia coli and by an in vivo rescue assay by expressing the mutant AceA in a Xanthomonas campestris gumH(-) strain. We found that only mutants K211A and E287A lost all detectable activity both in vitro and in vivo, whereas E295A retained residual activity in the more sensitive in vivo assay. H127A and S162A each retained reduced but significant activities both in vitro and in vivo. Secondary structure predictions of AceA and subsequent comparison with the crystal structures of the T4 beta-glucosyltransferase and MurG suggest that AceA Lys-211 and Glu-295 are involved in nucleotide sugar donor binding, leaving Glu-287 of the EX(7)E as a potential catalytic residue. (+info)
Characterization of a major cluster of nif, fix, and associated genes in a sugarcane endophyte, Acetobacter diazotrophicus.
A major 30.5-kb cluster of nif and associated genes of Acetobacter diazotrophicus (syn. Gluconacetobacter diazotrophicus), a nitrogen-fixing endophyte of sugarcane, was sequenced and analyzed. This cluster represents the largest assembly of contiguous nif-fix and associated genes so far characterized in any diazotrophic bacterial species. Northern blots and promoter sequence analysis indicated that the genes are organized into eight transcriptional units. The overall arrangement of genes is most like that of the nif-fix cluster in Azospirillum brasilense, while the individual gene products are more similar to those in species of Rhizobiaceae or in Rhodobacter capsulatus. (+info)
Acetic acid bacteria have been isolated from submerged high-acid spirit vinegar fermentations in the Southern part of Germany. Four strains (LTH 4560T, LTH 4341, LTH 4551 and LTH 4637) were characterized in more detail and it was revealed that they have in common certain properties such as requirement of acetic acid, ethanol and glucose for growth, and no over-oxidation of acetate. Growth occurs only at total concentrations (sum of acetic acid and ethanol) exceeding 6.0%. A method for their preservation was developed. Comparative analysis of the 16S rRNA revealed sequence similarities of >99% between strain LTH 4560T and the type strains of the related species Gluconacetobacter hansenii. However, low levels of DNA relatedness (<41 %) were determined in DNA-DNA similarity studies. In addition, specific physiological characteristics permitted a clear identification of the strains within established species of acetic acid bacteria. The strains could also be differentiated on the basis of the distribution of IS element 1031 C within the chromosome. Based on these results, the new species Gluconacetobacter entanii sp. nov. is proposed for strain LTH 4560T ( = DSM 13536T). A 16S-rRNA-targeted oligonucleotide probe was constructed that was specific for G. entanii, and the phylogenetic position of the new species was derived from a 16S-rRNA-based tree. (+info)
Transfer of Acetobacter oboediens Sokollek et al 1998 and Acetobacter intermedius Boesch et al. 1998 to the genus Gluconacetobacter as Gluconacetobacter oboediens comb. nov. and Gluconacetobacter intermedius comb. nov.
Acetobacter oboediens Sokollek et al. 1998 and Acetobacter intermedius Boesch et al. 1998 are transferred to the genus Gluconacetobacter as Gluconacetobacter oboediens comb. nov. and Gluconacetobacter intermedius comb. nov. because, on the basis of their 16S rRNA gene sequences, the type strains of both species are located in the cluster of the genus Gluconacetobacter along with those of Gluconacetobacter xylinus, Gluconacetobacter europaeus, Gluconacetobacter hansenii, Gluconacetobacter liquefaciens (the type species) and Gluconacetobacter diazotrophicus. The significance of growth on mannitol agar and the presence of a ubiquinone isoprenologue composed of Q-10 is discussed for characterization of the genus Gluconacetobacter. (+info)
Characterization of acetic acid bacteria in traditional acetic acid fermentation of rice vinegar (komesu) and unpolished rice vinegar (kurosu) produced in Japan.
Bacterial strains were isolated from samples of Japanese rice vinegar (komesu) and unpolished rice vinegar (kurosu) fermented by the traditional static method. Fermentations have never been inoculated with a pure culture since they were started in 1907. A total of 178 isolates were divided into groups A and B on the basis of enterobacterial repetitive intergenic consensus-PCR and random amplified polymorphic DNA fingerprinting analyses. The 16S ribosomal DNA sequences of strains belonging to each group showed similarities of more than 99% with Acetobacter pasteurianus. Group A strains overwhelmingly dominated all stages of fermentation of both types of vinegar. Our results indicate that appropriate strains of acetic acid bacteria have spontaneously established almost pure cultures during nearly a century of komesu and kurosu fermentation. (+info)