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The structural characterization of the lipopolysaccharide (LPS) from extremophiles has important implications in several biomedical and therapeutic applications. The polyextremophile Gram-negative bacterium Halobacteroides lacunaris TB21, isolated from one of the most extreme habitats on our planet, the deep-sea hypersaline anoxic basin Thetis , represents a fascinating microorganism to investigate in terms of its LPS component. Here we report the elucidation of the full structure of the R-type LPS isolated from H. lacunaris TB21 that was attained through a multi-technique approach comprising chemical analyses, NMR spectroscopy, and Matrix-Assisted Laser Desorption Ionization (MALDI) mass spectrometry. Furthermore, cellular immunology studies were executed on the pure R-LPS revealing a very interesting effect on human innate immunity as an inhibitor of the toxic Escherichia coli LPS., Competing Interests: The authors declare no conflict of interest.

Aims: (i) To investigate the enzymatic characterization of α-L-arabinofuranosidase from Thermotoga thermarum DSM5069. (ii) To evaluate the performance of its excellent properties on converting ginsenoside Rc to ginsenoside Rd., Methods and Results: The thermostable α-L-arabinofuranosidase (Tt-Afs) gene from T. thermarum DSM5069 was cloned and overexpressed. Recombinant Tt-Afs was purified, and its molecular weight was approx. 55 kDa. Its optimal activity was at pH 5·0 and 95°C. It has high selectivity for cleaving the outer arabinofuranosyl moieties at the C-20 carbon of ginsenoside Rc and its sugar-tolerance makes Tt-Afs a promising candidate for the production of ginsenoside Rd. In a reaction at 85°C and pH 5·0, 25 g l(-1) of ginsenoside Rc was transformed into 21·8 g l(-1) of Rd within 60 min, with a corresponding molar conversion of 99·4% and a high ginsenoside Rd productivity of 21800 mg l(-1) h(-1)., Conclusions: We have successfully cloned and overexpressed the novel α-l-arabinofuranosidase from T. thermarum DSM5069. The high ginsenoside Rd productivity and detailed characterization of recombinant Tt-Afs was provided., Significance and Impact of the Study: The result shows a high productivity on the bioconversion from high concentration ginsenoside Rc to ginsenoside Rd, which also give rise to a potential commercial enzyme application., (© 2016 The Society for Applied Microbiology.)

A strictly anaerobic, propionate-producing bacterial strain (WB4T) isolated from rice plant residue in anoxic rice-field soil in Japan was characterized phenotypically and phylogenetically. Cells were Gram-negative, non-motile, non-spore-forming, short rods. The strain utilized various sugars and produced propionate and acetate as major fermentation products with a small amount of succinate. The optimum growth temperature was 30 degrees C. Oxidase, catalase and nitrate-reducing activities were negative. The major cellular fatty acids were anteiso-C(15 : 0), C(15 : 0) and anteiso-C(17 : 0) 3-OH. Menaquinone MK-8(H4) was the major respiratory quinone. The genomic DNA G+C content was 39.3 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence placed the strain in the phylum 'Bacteroidetes'. The closest relative to strain WB4T was an environmental clone from water contaminated with equine manure (sequence similarity of 99.7 %) and the strain formed a distinct cluster with other environmental clones mainly from freshwater sediments. The closest recognized species were members of the genus Dysgonomonas, with 16S rRNA gene sequence similarities of 90.9-89.8 %. Bacteroides merdae was the next closest recognized species (similarity of 88.7 % to the type strain). Given that the ecological, physiological and chemotaxonomic characteristics of strain WB4T were different from those of any related species, a new genus and species Paludibacter propionicigenes gen. nov., sp. nov., is proposed to accommodate it. The type strain is WB4T (= JCM 13257T = DSM 17365T).

The influence of temperature on cellular fatty acid composition and on heat stress tolerance was studied in the two species of Pectinatus, an anaerobic gram-negative bacterium. Cellular fatty acid (FA) patterns were determined for Pectinatus species cultivated in MRS medium at various defined conditions of temperature and pH. Our study shows that fluctuations of growth temperature and pH induced important changes in the ratio of unsaturated FAs (UFAs) to saturated FAs (SFAs). The major differences in the FA composition as a function of growth temperature concerned C15:0 and C17:0 for the SFAs and C15:1 and C17:1 for the UFAs. The most significant adaptation of lipid composition to lower growth temperatures was the strong increase of UFAs, particularly for C15:1 and C17:1 concomitantly with a decrease of SFAs (C15:0 and C17:0). When the pH of the culture medium was lowered from 6.2 to 4.0, a notable drop in the synthesis of the UFAs C15:1 and C17:1 was observed together with an important increase of C18-cyclopropane (C18-cyc) and high carbon number SFAs. Thermal modifications also provoked changes in Pectinatus behaviour. We observed that P. cerevisiiphilus was more heat sensitive than P. frisingensis. Mild exponential phase cells were treated for 1 h, at 40 degrees C for P. cerevisiiphilus or at 41 degrees C for P. frisingensis. This thermal adaptation induced tolerance against heat challenge (49 and 50 degrees C for P. cerevisiiphilus and P. frisingensis, respectively). Survival of P. cerevisiiphilus and P. frisingensis adapted cells was, respectively, 3400- and 790-fold higher than control. Interestingly, adapted cells of P. cerevisiiphilus were more thermotolerant than P. frisingensis pretreated cells.

Spermidine and cadaverine were found to be constituents of the cell wall peptidoglycan of Anaerovibrio lipolytica, a strictly anaerobic bacterium. The peptidoglycan was degraded with the N-acetylmuramyl-L-alanine amidase and endopeptidase into two peptide fragments, peptide I and peptide II, at a molar ratio of 4:1. Peptides I and II were identified as L-alanine-D-glutamic acid(alphacadaverine)gammameso-diaminopimelic acid (DAP)-D-alanine and L-alanine-D-glutamic acid(alphaspermidine)gammameso-DAP-D-alanine, respectively. The N(1)-amino group of spermidine was linked to the alpha-carboxyl group of the D-glutamic acid residue of peptide II.

A novel cellulosomal scaffoldin gene, termed cipV, was identified and sequenced from the mesophilic cellulolytic anaerobe Acetivibrio cellulolyticus. Initial identification of the protein was based on a combination of properties, including its high molecular weight, cellulose-binding activity, glycoprotein nature, and immuno-cross-reactivity with the cellulosomal scaffoldin of Clostridium thermocellum. The cipV gene is 5,748 bp in length and encodes a 1,915-residue polypeptide with a calculated molecular weight of 199,496. CipV contains an N-terminal signal peptide, seven type I cohesin domains, an internal family III cellulose-binding domain (CBD), and an X2 module of unknown function in tandem with a type II dockerin domain at the C terminus. Surprisingly, CipV also possesses at its N terminus a catalytic module that belongs to the family 9 glycosyl hydrolases. Sequence analysis indicated the following. (i) The repeating cohesin domains are very similar to each other, ranging between 70 and 90% identity, and they also have about 30 to 40% homology with each of the other known type I scaffoldin cohesins. (ii) The internal CBD belongs to family III but differs from other known scaffoldin CBDs by the omission of a 9-residue stretch that constitutes a characteristic loop previously associated with the scaffoldins. (iii) The C-terminal type II dockerin domain is only the second such domain to have been discovered; its predicted "recognition codes" differ from those proposed for the other known dockerins. The putative calcium-binding loop includes an unusual insert, lacking in all the known type I and type II dockerins. (iv) The X2 module has about 60% sequence homology with that of C. thermocellum and appears at the same position in the scaffoldin. (v) Unlike the other known family 9 catalytic modules of bacterial origin, the CipV catalytic module is not accompanied by a flanking helper module, e.g., an adjacent family IIIc CBD or an immunoglobulin-like domain. Comparative sequence analysis of the CipV functional modules with those of the previously sequenced scaffoldins provides new insight into the structural arrangement and phylogeny of this intriguing family of microbial proteins. The modular organization of CipV is reminiscent of that of the CipA scaffoldin from C. thermocellum as opposed to the known scaffoldins from the mesophilic clostridia. The phylogenetic relationship of the different functional modules appears to indicate that the evolution of the scaffoldins reflects a collection of independent events and mechanisms whereby individual modules and other constituents are incorporated into the scaffoldin gene from different microbial sources.

Chemotaxonomic, electron microscopic and 16S rRNA gene sequence analyses of the three described species of the genus Anaerovibrio demonstrated only remote similarities to each other. The 16S rRNA gene sequence similarities between Anaerovibrio lipolytica, Anaerovibrio glycerini and Anaerovibrio burkinabensis and the derived phylogenetic relationships of the three species studied fell below genus level. All three species clustered within the Sporomusa-Pectinatus-Selenomonas phyletic group. Each species showed a distinct phospholipid pattern and whole-cell fatty acid distribution. Several isoprenologues of the lipoquinone 'lipid F' were found to differ in their quantitative distribution in the Anaerovibrio species. On the basis of these results, the new genera Anaerosinus gen. nov. and Anaeroarcus gen. nov. are proposed. The type species of Anaerosinus is Anaerosinus glycerini comb. nov., and the type species of Anaeroarcus is Anaeroarcus burkinensis [corrig.] comb. nov. The genus Anaerovibrio is consequently restricted to a single species, namely Anaerovibrio lipolyticus [corrig.]

A soluble c-type cytochrome was first purified from Geobacter metallireducens to an electrophoretically homogeneous state. The purified cytochrome c showed absorption peaks at 530 and 409 nm in the oxidized form and 552, 522, and 418 nm in the reduced form. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate allowed us to calculate the molecular mass at 9.5 kDa. It contained 3 mol of heme c per molecule of the protein on the basis of heme c and protein concentration. The mid-point redox potential at pH 7.0 was determined to be -190 mV. Although the N-terminal amino acid sequence of the first 17 residues was similar to that of Desulfuromonas acetoxidans cytochrome c7, G. metallireducens cytochrome c did not show Fe(III)-reducing activity.

This study was conducted to investigate the effects of sonicated bacterial extracts (SBEs) from anaerobic Gram-negative bacteria on periapical fibroblast obtained from the apical portion of human periodontal ligaments. Porphyromonas endodontalis, Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum were chosen from among the endodontic bacteria isolated from root canals having a periapical lesion and compared in terms of their cytotoxicity. The purpose of this study was to examine which bacteria are involved in the development of periapical inflammation. The anaerobes were cultured under strict anaerobic conditions, and the bacterial cells were then harvested by centrifugation after incubation. The concentrated cell suspensions were sonicated and subsequently centrifuged. An SBE was made of each of the filtered supernatants. Each SBE was added to cultures of periapical fibroblasts. The cell growth and proliferation were measured by the MTT method after 3, 5, and 7 days. The SBEs from P. endodontalis, P. gingivalis, and F. nucleatum inhibited the growth of the fibroblasts, whereas the SBE from P. intermedia did not inhibit it. The SBEs from P. gingivalis and F. nucleatum inhibited the fibroblast growth more strongly than did the P. endodontalis, P. gingivalis, and F. nucleatum may participate in the development of periapical lesions.

A thermophilic, anaerobic, strictly autotrophic, sulphur-reducing bacterium, designated BSAT (T = type strain), was isolated from a deep-sea hydrothermal chimney sample collected at the mid-Atlantic ridge. Gram-negative cells occurred singly or in pairs as small highly motile rods. Spores were not observed. The temperature range for growth was 40 to 75 degrees C, with an optimum at 70 degrees C. The pH range for growth at 70 degrees C was from 4.4 to 7.5, with an optimum around 6.0. The sea salt concentration range for growth was 15-70 gI(-1) with an optimum at 35 gI(-1). Elemental sulphur, thiosulphate and sulphite were reduced to hydrogen sulphide. Sulphate and cystine were not reduced. The G+C content of the genomic DNA was 35 mol%. Phylogenetic analyses of the 16S rRNA gene indicated that the strain was a member of the domain Bacteria and formed a branch that was almost equidistant from members of the orders Aquificales and Thermotogales. The new organism possesses phenotypic and phylogenetic traits that do not allow its classification as a member of any previously described genus; therefore, it is proposed that this isolate should be described as a member of a novel species of a new genus, Desulfurobacterium gen. nov., of which Desulfurobacterium thermolithotrophum sp. nov. is the type species. The type strain is BSAT (= DMS 11699T).

Thioredoxin reductase and thioredoxin are primarily involved in catabolic metabolism as important electron carriers in anaerobic, amino-acid-degrading bacteria. A general and fast procedure was developed for the purification of thioredoxin reductase and thioredoxin from Eubacterium acidaminophilum, Clostridium litorale, C. sticklandii, C. sporogenes, C. cylindrosporum and 'Tissierella creatinophila' based upon their properties: the binding to 2',5'-AMP-Sepharose by thioredoxin reductase and the inability of thioredoxins to bind to a DEAE-Sephacel column. The consensus sequence at the active site of thioredoxins (-WCGPC-) was found to be modified in all of these anaerobes: Trp-31 (Escherichia coli nomenclature) was replaced by Gly or Ser, Gly-33 by Val or Glu. None of these thioredoxins reacted with thioredoxin reductase of E. coli or vice versa, but they did interact with the thioredoxin reductases obtained from the other anaerobes studied. Based upon their distinguishing features it is suggested that these thioredoxins might form an evolutionarily separate group.

The crystal structure of CheY protein from Thermotoga maritima has been determined in four crystal forms with and without Mg++ bound, at up to 1.9 A resolution. Structural comparisons with CheY from Escherichia coli shows substantial similarity in their folds, with some concerted changes propagating away from the active site that suggest how phosphorylated CheY, a signal transduction protein in bacterial chemotaxis, is recognized by its targets. A highly conserved segment of the protein (the "y-turn loop," residues 55-61), previously suggested to be a rigid recognition determinant, is for the first time seen in two alternative conformations in the different crystal structures. Although CheY from Thermotoga has much higher thermal stability than its mesophilic counterparts, comparison of structural features previously proposed to enhance thermostability such as hydrogen bonds, ion pairs, compactness, and hydrophobic surface burial would not suggest it to be so.

During our studies of the bacterial etiology of appendicitis, we often isolated a previously undescribed anaerobic gram-negative rod. This organism resembled the Bacteroides fragilis group because it was resistant to bile and because of its special-potency-disk pattern (resistant to vancomycin, kanamycin, and colistin), but unlike the B. fragilis group, this bacterium produced brown pigment on media containing hemolysed blood. The cellular fatty acid pattern, with iso-C15:0 being the predominant acid, was most closely related to the fatty acid profile of Porphyromonas species; however, this organism differed from Porphyromonas species by being bile-resistant and by not producing butyrate as a metabolic endproduct. Enzymatic activities of 31 isolates were determined with use of the API ZYM system and Rosco diagnostic tablets. These profiles were different from those of Prevotella, Porphyromonas, and related species. This organism was isolated from 40% of appendiceal tissue samples; no obvious qualitative or quantitative difference in rates of isolation from patients with inflamed or normal appendices was observed.

The fatty acid compositions of the hyperthermophilic microorganisms Thermotoga maritima and Pyrococcus furiosus were studied and compared. A total of 37 different fatty acids were identified in T. maritima, including the novel 13,14-dimethyloctacosanedioic acid. In contrast, a total of 18 different fatty acids were characterized, as minor components, in P. furiosus, and these included saturated, monounsaturated, and dicarboxylic acids. This is the first report of fatty acids from an archaeon.

Understanding the molecular mechanisms behind extreme temperature stability is of relevance for the protein folding problem and for designing proteins for industrial and medical applications. A powerful approach for understanding the structural basis of thermostability is the comparison of high resolution structures of homologous proteins from mesophiles and thermophiles. The 1.75 A crystal structure of Thermotoga maritima 1[4Fe-4S] ferredoxin was compared with those of mesophilic ferredoxins. Detailed analysis of structural differences reveals that thermostability is achieved without large changes of the overall polypeptide chain folding. The most striking differences include the formation of additional hydrogen bonding networks involving both side-chain and main-chain atoms. These networks are mainly connecting turns and strongly fix the N-terminus to the central core of the protein, increasing the overall rigidity of Thermotoga maritima ferredoxin. Other possibly stabilizing factors are the shortening of a solvent exposed surface loop, the increased content of alanines in the second alpha-helix, and the replacement of three residues close to the iron-sulfur cluster, which are in energetically unfavourable conformations in other ferredoxins, by glycines.

Forty-nine isolates of Butyrivibrio fibrisolvens and a single isolate of Butyrivibrio crossotus were screened for the production of inhibitors by a deferred plating procedure. Twenty-five isolates produced factors which, to various degrees, inhibited the growth of the other Butyrivibrio isolates. None of the inhibitory activity was due to bacteriophages. The inhibitory products from 18 of the producing strains were sensitive to protease digestion. Differences in the ranges of activity among the Butyrivibrio isolates and protease sensitivity profiles suggest that a number of different inhibitory compounds are produced. These findings suggest that the production of bacteriocin-like inhibitors may be a widespread characteristic throughout the genus Butyrivibrio. The bacteriocin-like activity from one isolate, B. fibrisolvens AR10, was purified and confirmed to reside in a single peptide. Crude bacteriocin extracts were prepared by ammonium sulfate and methanol precipitation of spent culture supernatants, followed by dialysis and high-speed centrifugation. The active component was isolated from the semicrude extract by reverse-phase chromatography. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed that the peptide was purified to homogeneity, having an estimated molecular mass of approximately 4,000 Da. The N terminus of the peptide was blocked. A cyanogen bromide cleavage fragment of the native peptide yielded a sequence of 20 amino acids [(M)GIQLAPAXYQDIVNXVAAG]. No homology with previously reported bacteriocins was found. Butyrivibriocin AR10 represents the first bacteriocin isolated from a ruminal anaerobe.

Four strains of gram-negative, anaerobic, non-spore-forming bacteria that were curved rods which were motile by means of flagella originating from the concave side of the cells and which fermented succinate quantitatively to propionate were isolated from high dilutions of rumen ingesta obtained from cows on pasture. The bacteria were asaccharolytic and not proteolytic and did not ferment amino acids or peptides. Succinate was the only substrate fermented. Rumen fluid together with yeast extract was required for good growth on succinate. Growth on succinate was enhanced in the presence of fumarate. The strains did not grow at 22 degrees C, and growth at 45 degrees C was in all cases less than growth at 39 degrees C. The cellular fatty acid compositions of all four strains were determined. The DNA base composition was about 46 mol% G + C. The complete 16S ribosomal DNA sequence of the type strain (strain S1-1) was determined, and the phylogenetic relationships were analyzed. The most closely related genera were the genera Selenomonas, Zymophilus, and Pectinatus, whereas the recently described succinate-fermenting organism Succiniclasticum ruminis was distantly related. The name proposed for these strains is Schwartzia succinivorans gen. nov., sp. nov.; the type strain is strain S1-1 (= DSM 10502). These organisms are common inhabitants of the rumina of cows on pasture.

The aim of this work was to test the feasibility of introducing an anaerobic microbial reductive dechlorination activity into non sterile soil slurry microcosms by inoculation with the pure anaerobic bacterial strain Desulfomonile tiedjei, which is capable of dechlorinating 3-chlorobenzoate to benzoate. To show that the bacterium was established in the microcosms we followed the expression of the reductive dechlorination activity and a molecular probe based on PCR amplification of the 16S rDNA gene was developed. However, the success of PCR amplification of the 16S rDNA gene depends on the DNA extraction and purification methodologies applied, as shown through the use of several protocols. In this study we report a DNA extraction and purification method which generates sufficient and very clean DNA suitable for PCR amplification of the D. tiedjei 16S rDNA gene. The threshold of detection was about 5.10(3) bacteria per gram of soil slurry. Introduction of D. tiedjei in soil slurry microcosms proved successful since 3-chlorobenzoate dechlorination activity was established with this bacterium in microcosms normally devoid of this dechlorination capacity. Indeed, the addition of D. tiedjei to microcosms supplemented with acetate plus formate as cosubstrate, at their respective concentrations of 5 and 6 mM, led to a total biotransformation of 2.5 mM of 3-chlorobenzoate within 12 days. After complete 3-chlorobenzoate dechlorination, the 16S rDNA gene of this bacterium was specifically detected only in the inoculated microcosms as shown by PCR amplification followed by restriction mapping confirmation.

Background: The characterization of the structural features that account for the high thermostability of some proteins is of great scientific and biotechnological interest. Proteins from hyperthermophilic organisms with optimum growth temperatures of 80 degrees C and higher generally show high intrinsic stabilities. The comparison of high resolution X-ray structures of these proteins with their counterparts from mesophilic organisms has therefore helped to identify potentially stabilizing forces in a number of cases. Small monomeric proteins which comprise only a single domain, such as ferredoxins, are especially suitable for such comparisons since the search for determinants of protein stability is considerably simplified., Results: The 1.75 A crystal structure of the extremely thermostable 1[4Fe-4S] ferredoxin from Thermotoga maritima (FdTm) was determined and compared with other monocluster-containing ferredoxins with different degrees of thermostability., Conclusions: A comparison of the three-dimensional structure of FdTm with that of ferredoxins from mesophilic organisms suggests that the very high thermostability of FdTm is unexpectedly achieved without large changes of the overall protein structure. Instead, an increased number of potentially stabilizing features is observed in FdTm, compared with mesophilic ferredoxins. These include stabilization of alpha helices, replacement of residues in strained conformation by glycines, strong docking of the N-terminal methionine and an overall increase in the number of hydrogen bonds. Most of these features stabilize several secondary structure elements and improve the overall rigidity of the polypeptide backbone. The decreased flexibility will certainly play a relevant role in shielding the iron-sulfur cluster against physiologically high temperatures and further improve the functional integrity of FdTm.

The accumulation of intracellular organic solutes was examined in six species of the order Thermotogales by nuclear magnetic resonance spectroscopy. The newly discovered compounds di-2-O-beta-mannosyl-di-myo-inositol-1,1'(3,3')-phosphate and di-myo-inositol-1,3'-phosphate were identified in Thermotoga maritima and Thermotoga neapolitana. In the latter species, at the optimum temperature and salinity the organic solute pool was composed of di-myo-inositol-1,1'(3,3')-phosphate, beta-glutamate, and alpha-glutamate in addition to di-myo-inositol-1,3'-phosphate and di-2-O-beta-mannosyl-di-myo-inositol-1,1'(3,3')-phosphate. The concentrations of the last two solutes increased dramatically at supraoptimal growth temperatures, whereas beta-glutamate increased mainly in response to a salinity stress. Nevertheless, di-myo-inositol-1,1'(3,3')-phosphate was the major compatible solute at salinities above the optimum for growth. The amino acids alpha-glutamate and proline were identified under optimum growth conditions in Thermosipho africanus, and beta-mannosylglycerate, trehalose, and glycine betaine were detected in Petrotoga miotherma. Organic solutes were not detected, under optimum growth conditions, in Thermotoga thermarum and Fervidobacterium islandicum, which have a low salt requirement or none.

In vitro experiments with 3,4-dimethoxybenzoate-induced Sporomusa enzymes a broad O-methyl ether cleavage capacity. The O-demethylase activity hydrolized the methyl-oxygen linkages of methoxynaphtholes of the heterocycles 2-methoxyfuran or 2-methoxythiophene as well as of several dimethoxy and monomethoxy aryls under anaerobic conditions. Also, fluoro and chloro substituents of anisoles enhanced the O-demethylation rate, indicating that an electron delocalized aromatic structure supported the methyl ether activation mechanism. Monomethoxy aromatics with additional chargeable groups, however, were less effectively transformed by the O-demethylase activity. No transformations into hydroxylated products occurred with 4-(trifluoromethoxy)benzyl alcohol, 4-(trifluoromethoxy)fluorobenzene, 2,5-dimethoxytetrahydrofuran, or alkyl-O-methyl ethers. The inert ethers did not affect the 3,4-dimethoxybenzoate metabolism. Ether activation or the following methyl transfer to the methyl acceptor tetrahydrofolate involved a prominent 31 kDa peptide from the cytoplasmic cell fraction, because this particular peptide was lacking in cells grown with methanol, betaine or fructose.

The solution structure of the 60-residue 1[Fe4-S4] ferredoxin from the hyperthermophilic bacterium Thermotoga maritima was determined based on 683 distance and 35 dihedral angle restraints that were obtained from NMR data. In addition, data known from crystallographic studies of ferredoxins was used for modeling of the iron-sulfur cluster and its environment. The protein shows a globular fold very similar to the fold of the related 1[Fe4-S4] ferredoxins from Desulfovibrio gigas and Desulfovibrio africanus, and elements of regular secondary structure similar to those in other ferredoxins were found in the T. maritima protein. In particular, the T. maritima protein displayed a beta-sheet structure made up of strands located at the very NH(2) and COOH termini of the protein, and an internal alpha-helix. The internal beta-sheet observed in the D. gigas and D. africanus ferredoxins could not be confirmed in T. maritima ferredoxin and is thus suggested to be only weakly present or even absent in this protein. This result suggests that thermostability in ferredoxins is not necessarily correlated with the content of stable elements of regular secondary structure.

The structure of Butyrivibrio fibrisolvens strain 49 capsular polysaccharide has been investigated mainly by sugar and methylation analysis, partial chemical degradations, NMR spectroscopy, and mass spectrometry. The results suggest that the polysaccharide is composed of pentasaccharide repeating units having the following structure. [formula: see text] The polysaccharide contains O-acetyl groups, one of which is substituted to O-3 of the 4-substituted alpha-D-Galp residue, while others occur in non-stoichiometric amounts at other locations.

The hyperthermophilic bacterium Thermotoga maritima and the hyperthermophilic archaeon Pyrococcus furiosus grow optimally at 80 and 100 degrees C, respectively, by the fermentation of carbohydrates to organic acids, CO2, and H2. Pyruvate is a major source of reductant for H2 production during fermentation, and pyruvate ferredoxin oxidoreductase (POR), a 4Fe-type ferredoxin, and hydrogenase have been previously purified from both species. P. furiosus utilizes a copper-iron-containing POR and a nickel-iron-containing hydrogenase, whereas the POR of T. maritima lacks copper and its hydrogenase lacks nickel. For all four enzymes and for the two ferredoxins, we have determined their reduction potentials (E degrees') and, where possible, thermodynamic parameters associated with electron transfer (delta S degrees and delta H degrees), using differential pulse voltammetry at temperatures ranging from 25 to 95 degrees C. At ambient temperature, the E degrees' values for all six proteins were comparable and spanned less than 50 mV, but their temperature dependence varied dramatically, even between analogous proteins, such that in the physiological-relevant temperature range the E degrees' values became widely separated. In most cases, transition points were observed in E degrees'/temperature profiles, and these generally corresponded with significant increases in catalytic activity, but occurred at lower temperatures in T. maritima than in P. furiosus. The two ferredoxins (and also P. furiosus rubredoxin) had much more negative entropy terms than were calculated for POR and hydrogenase, and these values were also more negative than those previously reported for mesophilic redox proteins. The reduction potentials measured at high temperatures and likely efficiencies of electron transfer between the various proteins were consistent with in vitro activity measurements.(ABSTRACT TRUNCATED AT 250 WORDS)

The oxidized Fe4S4 ferredoxin from the hyperthermophilic bacterium Thermotoga maritima has been investigated by one- and two-dimensional NMR in order to characterize its hyperfine-shifted resonances originating from the cysteinyl cluster ligands and to assign its resonances in the diamagnetic shift range. The chemical shift and relaxation time pattern of the hyperfine-shifted signals is very similar to other oxidized Fe4S4 ferredoxins. A tentative sequence-specific assignment of these resonances according to a general pattern of chemical shift of cysteine protons versus sequence position of cluster ligand is presented. Furthermore, sequence-specific assignments for 85% of the amino acid residues that were obtained without any guidance by known X-ray structures of ferredoxins are given. They reveal the formation of at least two elements of secondary structure by the polypeptide chain of T. maritima ferredoxin: an alpha-helix comprising residues C43-D49 and a double-stranded antiparallel beta-sheet consisting of the N- and C-terminal parts of the protein. This folding pattern is very similar to that of the crystallographically characterized ferredoxin from the mesophile Desulfovibrio gigas [Kissinger, C.R., Sieker, L.C., Adman E.T. & Jensen, L.H. (1991) J. Mol. Biol. 219, 693-715] and therefore suggesting different mechanisms of stabilization for T. maritima ferredoxin and the ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus that was recently investigated by NMR [Teng, Q., Zhou, Z.H., Smith, E.T., Busse, S. C., Howard, J.B., Adams M.W.W. & La Mar, G.N. (1994) Biochemistry 33, 6316-6326].

Objectives: To compare the osteolytic activity of surface-associated material (SAM) and lipid A-associated proteins (LAPs) from periodontopathogenic bacteria., Materials and Methods: Surface-associated material was extracted from the surface and LAPs from the cell walls of a range of periodontopathic bacteria including Actinobacillus actinomycetemcomitans and Eikenella corrodens. These bacterial fractions were assayed to determine their composition and their capacity to induce bone resorption was determined by use of the neonatal murine calvarial bone resorption assay., Results: The SAMs from E. corrodens and A. actinomycetemcomitans demonstrated bone-resorbing capacity at concentrations as low as 1 ng ml-1 which, given the molecular weights of the active components, is in the picomolar range of activity. In contrast, the SAMs from the other three bacteria were significantly less potent and showed a lower efficacy. The LAPs all showed significant, and similar, capacities to induce bone breakdown., Conclusions: This is the first demonstration that LAP from periodontopathic bacteria can stimulate bone degradation. The LAPs from diverse bacteria all produced similar levels of bone-resorbing activity. In contrast, the SAM showed significant differences in potency and in efficacy (maximal stimulation). This may mean that in vivo certain periodontopathic bacteria have significantly more bone-resorbing capacity than others and should be therapeutic targets.

A ferredoxin has been purified from one of the most ancient and most thermophilic bacteria known, Thermotoga maritima, which grows up to 90 degrees C. The reduced protein (M(r) approx. 6300) contains a single S = 1/2 [4Fe-4S]1+ cluster with complete cysteinyl ligation, and was unaffected after incubation at 95 degrees C for 12 h. It functioned as an electron carrier for T. maritima pyruvate oxidoreductase. Remarkably, the properties and amino acid sequence of this hyperthermophilic bacterial protein are much more similar to those of ferredoxins from hyperthermophilic archaea, rather than ferredoxins from mesophilic and moderately thermophilic bacteria.

Cellular fatty acid profiles were determined for species in 33 genera of anaerobic gram-negative bacilli and were confirmed to be a useful taxonomic tool. Most of the genera could be differentiated by visual inspection of their profiles. The three genus pairs that were most difficult to distinguish visually (Bacteroides and Prevotella, Pectinatus and Megamonas, and Serpulina and Bilophila) and the species of these genera were differentiated by the MIDI (Microbial ID, Inc.) identification system. Similarities in cellular fatty acid profiles may be correlated with similarities in other phenotypic characteristics, but more often there is no other obvious phenotypic relationship. Although medium components may not change the constituents detected or the ratios among the constituents detected for some species, identical medium changes may result in vast differences in the profiles obtained with other species. Thus, if a worker wishes to compare profiles of various taxa, it is essential that the same cultural and analytical conditions be used.

Marker components of the phospholipids of Ruminococcus flavefaciens and Fibrobacter succinogenes were identified for studies on the degradation of forage by these bacteria growing in mixed culture. The principal fatty acid methyl esters and dimethyl acetals detected varied between strains and were influenced by the addition of a mixture of higher volatile fatty acids and vitamins to the medium, but these effects were small compared to the differences between the species. When two strains of R. flavefaciens were grown on a mixture of clover and ryegrass, and on barley straw in the presence or absence of two strains of F. succinogenes, the solubilization of plant material tended to be lowered by the presence of F. succinogenes. R. flavefaciens was the predominant bacterium among colonies recovered from roll tubes, and the phospholipids were primarily those of R. flavefaciens. Analysis of the culture supernatant liquids showed that F. succinogenes produced greater amounts of free and bound xylose from both clover and straw than did R. flavefaciens. With both forages, cultures containing the two species produced more soluble free arabinose, and less soluble-bound arabinose, than either species grown alone.

The capsular polysaccharide from Butyrivibrio fibrisolvens strain X6C61 has been investigated using NMR spectroscopy, mass spectrometry, methylation analysis, and partial acid hydrolysis as the main methods. The polysaccharide is composed of hexasaccharide repeating units having the following structure. [formula: see text] The polysaccharide also contains O-acetyl groups, of which approximately 70% are substituted to O-3 of the beta-D-Glc pA residue.

A disaccharide containing GlcN and 3-deoxy-D-manno-2-octulosonic acid (Kdo) was detected after acidic methanolysis and peracetylation of hydrofluoric-acid-treated smooth-type and rough-type lipopolysaccharide of Pectinatus cerevisiiphilus and Pectinatus frisingensis, which are strictly anaerobic bacteria capable of growing in packaged beer. The disaccharide was also found in alkali-treated lipopolysaccharide, but was not directly detectable from intact lipopolysaccharide. This suggested that the disaccharide carried a phosphate residue. The position of this phosphate was shown, by GLC/MS of appropriately degraded and derivatized samples, to be O6 of the GlcN. Methylation analysis of the purified disaccharide revealed that GlcN was linked to position 4 of Kdo. The acetylated derivative of the disaccharide was isolated in pure form, and, by 1H-NMR and 13C-NMR spectroscopy, it was confirmed to possess the structure alpha-D-GlcpN-(1'-->4)-Kdo. In the lipopolysaccharide the amino group of GlcN is free.

The gram-negative metal-reducing microorganism, previously known as strain GS-15, was further characterized. This strict anaerobe oxidizes several short-chain fatty acids, alcohols, and monoaromatic compounds with Fe(III) as the sole electron acceptor. Furthermore, acetate is also oxidized with the reduction of Mn(IV), U(VI), and nitrate. In whole cell suspensions, the c-type cytochrome(s) of this organism was oxidized by physiological electron acceptors and also by gold, silver, mercury, and chromate. Menaquinone was recovered in concentrations comparable to those previously found in gram-negative sulfate reducers. Profiles of the phospholipid ester-linked fatty acids indicated that both the anaerobic desaturase and the branched pathways for fatty acid biosynthesis were operative. The organism contained three lipopolysaccharide hydroxy fatty acids which have not been previously reported in microorganisms, but have been observed in anaerobic freshwater sediments. The 16S rRNA sequence indicated that this organism belongs in the delta proteobacteria. Its closest known relative is Desulfuromonas acetoxidans. The name Geobacter metallireducens is proposed.

A recently isolated, selenate-respiring microorganism (strain AXT [T = type strain]) was classified by using a polyphasic approach in which both genotypic and phenotypic characteristics were determined. Strain AXT is a motile, gram-negative, rod-shaped organism with a single polar flagellum. On the basis of phenotypic characteristics, this organism can be classified as a Pseudomonas sp. However, a comparison of the 16S rRNA sequence of strain AXT with the sequences of other organisms indicated that strain AXT is most similar to members of the beta subclass (level of similarity, 86.8%) rather than to members of the gamma subclass (level of similarity, 80.2%) of the Proteobacteria. The presence of the specific polyamine 2-hydroxyputrescine and the presence of a ubiquinone with eight isoprenoid units in the side chain (ubiquinone Q-8) excluded strain AXT from the authentic genus Pseudomonas and allowed placement in the beta subclass of the Proteobacteria. Within the beta subclass, strain AXT is related to Iodobacter fluvatile. The phylogenetic distance (level of similarity, less than 90%), as well as a lack of common phenotypic characteristics between these organisms, prevents classification of strain AXT as a member of the genus Iodobacter. In addition, strain AXT possesses a unique mechanism for anaerobic respiration, which allows it to utilize selenate as an electron acceptor without interference by nitrate. Therefore, we propose that strain AXT should be the first member of a new genus and species, Thauera selenatis.

We have discovered a new oligomeric protein component associated with the outer membrane of the ancestral eubacterium Thermotoga maritima. In electron micrographs, the protein, Omp alpha, appears as a rod-shaped spacer that spans the periplasm, connecting the outer membrane to the inner cell body. Purification, biochemical characterization and sequencing of Omp alpha suggest that it is a homodimer composed of two subunits of 380 amino acids with a calculated M(r) of 43,000 and a pI of 4.54. The sequence of the omp alpha gene indicates a tripartite organization of the protein with a globular NH2-terminal domain of 64 residues followed by a putative coiled-coil segment of 300 residues and a COOH-terminal, membrane-spanning segment. The predicted length of the coiled-coil segment (45 nm) correlates closely with the spacing between the inner and outer membranes. Despite sequence similarity to a large number of coiled-coil proteins and high scores in a coiled-coil prediction algorithm, the sequence of the central rod-shaped domain of Omp alpha does not have the typical 3.5 periodicity of coiled-coil proteins but rather has a periodicity of 3.58 residues. Such a periodicity was also found in the central domain of staphylococcal M protein and beta-giardin and might be indicative of a subclass of fibrous proteins with packing interactions that are distinct from the ones seen in other two-stranded coiled-coils.

Bilophila wadsworthia, a recently described anaerobic gram-negative bacillus, was isolated from blood cultures of two patients with liver abscesses. These isolates exhibited the expected phenotypic characteristics, and identification was confirmed by cell wall fatty acid analysis by gas-liquid chromatography. This is the first documentation of bacteremia due to B. wadsworthia.

Clinical (66) and reference (5) strains of pigmented gram-negative anaerobic bacilli, identified as Prevotella intermedia (47), Pr. melaninogenica (1), Pr. corpora (8), Porphyromonas asaccharolyticus (12), P. endodontalis (1) and P. gingivalis (2), were examined by pyrolysis mass spectrometry (PMS) and in conventional tests. Numerical classification based on conventional test reaction patterns (CTRPs) resolved five clusters, four comprising strains identified as Pr. intermedia, Pr. corpora, Pr. melaninogenica, and P. gingivalis respectively, and one comprising strains identified as P. asaccharolyticus and P. endodontalis. Numerical classification based on PMS showed a similar division, with decreasing homogeneity of chemical composition in the order Pr. intermedia, Pr. corpora, P. asaccharolyticus, which agreed with the order of homogeneity in CTRPs. PMS clusters corresponding to the genus Porphyromonas were clearly distinct from those of the genus Prevotella. PMS and CTRP classification disagreed on cluster membership for six strains. PMS identification from blind challenge sets was in agreement with conventional identification for 64 of 67 strains.

We had proposed earlier that the anaerobe Oxalobacter formigenes sustains a proton-motive force by exploiting a secondary carrier rather than a primary proton pump. In this view, a carrier protein would catalyze the exchange of extracellular oxalate, a divalent anion, and intracellular formate, the monovalent product of oxalate decarboxylation. Such an electrogenic exchange develops an internally negative membrane potential, and since the decarboxylation reaction consumes an internal proton, the combined activity of the carrier and the soluble decarboxylase would constitute an "indirect" proton pump with a stoichiometry of 1H+ per turnover. This model is now verified by identification and purification of OxlT, the protein responsible for the anion exchange reaction. Membranes of O. formigenes were solubilized at pH 7 with 1.25% octyl glucoside in 20 mM 3-(N-morpholino)propanesulfonic acid/K, in the presence of 0.4% Escherichia coli phospholipids and with 20% glucerol present as the osmolyte stabilant. Rapid methods for reconstitution were developed to monitor the distribution of OxlT during biochemical fractionation, allowing its purification by sequential anion and cation exchange chromatography. OxlT proved to be a single hydrophobic polypeptide, of 38 kDa mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a turnover number estimated as at least 1000/s. The properties of OxlT point to an indirect proton pump as the mechanism by which a proton-motive force arises in O. formigenes, and one may reasonably argue that indirect proton pumps take part in bacterial events such as acetogenesis, malolactate fermentation, and perhaps methanogenesis.

Lipopolysaccharides (LPS) from the type strains of the anaerobic beer spoilage bacteria Pectinatus cerevisiiphilus and P. frisingensis were extracted with the 5:5:8 volume ratio modification of the phenolchloroform-petroleum ether method (H. Brade and C. Galanos, Eur. J. Biochem. 122:233-237, 1982). Sequential precipitations of LPS with water and acetone from the phenol phase yielded LPS which differed in that water-precipitable material (LPS-H2O; 0.1 to 0.4% of the dry weight of the cells) was rough-type LPS, whereas acetone-precipitable material (LPS-Ac; 4.6 to 5.8% of the dry weight) contained both rough-type LPS and high-molecular-weight material resembling smooth LPS. The LPS were chemically characterized, and they contained D-glucosamine, 4-amino-4-deoxy-L-arabinose, 3-deoxy-D-manno-2-octulosonic acid, D-fucose, D-galactose, D-glucose, D-mannose, and phosphate. D-Fucose was present mostly in LPS-Ac, suggesting that it is a constituent of the O antigen. The major fatty acids were ester- and amide-linked (R)-3-hydroxytridecanoic and ester-linked undecanoic acids, with minor amounts of ester-linked tridecanoic and (R)-3-hydroxyundecanoic acids. The chemical compositions of LPS-H2O and LPS-Ac suggested that they differ not only in their smooth or rough nature but also in the structure of their core regions. This may explain their different precipitabilities from the extraction mixture. The extraction method was also shown to be applicable to the isolation of smooth-type LPS from Salmonella enterica serovar Typhimurium. Extraction of two Typhimurium strains carrying chemically different O antigens resulted in high yields (8% of the dry weight) of LPS. Strain SH2183, which contains the relatively hydrophobic O-4,5,12 antigen yielded almost exclusively LPS-Ac, whereas the LPS of strain SH5770, which has a hydrophilic O-6,7 antigen, was exclusively LPS-H2O. No fractionation to smooth and rough LPS occurred with the Typhimurium strains.

A numerical taxonomy was performed on 157 cultures (141 different strains) of species of Bacteroides, Polyphyromonas, Prevotella [not Prevotella (Labroue, 1976)] and Fusobacterium. Isolates were each tested for 111 phenotypic characters which included possession of constitutive enzymes, fermentation of specific carbohydrates, gas chromatographic analysis of metabolic end-products and of cellular carboxylic acid composition. Computation of similarity coefficients was followed by a single-linkage cluster analysis. At the 94% similarity level, the following groupings at genus level were apparent: (1) Bacteroides ureolyticus; (2) Fusobacterium mortiferum, F. necrogenes, F. necrophorum, F. nucleatum and F. varium; (3) B. caccae, B. distasonis, B. eggerthii, B. fragilis, B. merdae, B. ovatus, B stercoris, B. thetaiotaomicron, B. uniformus and B. vulgatus; (4) B. splanchnicus; (5) Porphyromonas asaccharolytica; (6) B. bivius (Prevotella bivia); (7) B. disiens (P. disiens); (8) B. intermedius (P. intermedia);and (9) B. melaninogenicus (P. melaninogenica). Single isolates of B. ruminicola (P. ruminicola), B. denticola (P. denticola) and B. capillosus did not cluster with other strains.

The major cobamide-containing protein from methanol-utilizing Sporomusa ovata was 8-fold enriched to apparent homogeneity. The protein exhibited a molecular mass of 40 kDa and of 38 kDa determined by gel filtration and by SDS-polyacrylamide gel electrophoresis, respectively. This finding indicates a monomeric protein structure. Monospecific polyclonal antisera raised against the protein did not cross react with another cobamide-containing protein from Sporomusa cells. Only the 40 kDa cobamide-containing protein was induced by methanol, since proteins from cells grown on 3,4-dimethoxybenzoate, betaine H2/CO2, or fructose showed faint or no cross reaction. Hence, the 40 kDa cobamide-containing protein is presumably involved in the methyl-transfer reaction of the methanol metabolism. The purified enzyme revealed 1.1 mol of p-cresolyl cobamide per mol of protein, but it lacked of iron-sulfur centers. Remarkably, the cofactor was firmly bound to its protein.

A commercially available, computer-assisted microbial identification system (MIS) employs gas-liquid chromatographic analysis of cellular fatty acids for bacterial identification. MIS was compared with conventional identification systems. Of 225 gram-negative anaerobes tested, MIS identified 72.4% of the strains to the species level, 88.9% to the appropriate group, and 93.3% to the correct genus.