Low-molecular weight keratins with anti-skin aging activity produced by anaerobic digestion of poultry feathers with Fervidobacterium islandicum AW-1.

Bioactive peptides contribute to various cellular processes including improved skin physiology. Hence, bioactive keratins have attracted considerable attention as active cosmetic ingredients for skin health. Here, we obtained low molecular weight (LMW) keratins from native chicken feathers by anaerobic digestion with an extremely thermophilic bacterium Fervidobacterium islandicum AW-1, followed by stepwise fractionation through ultrafiltration. To assess the effects of the feather keratins on skin health, we performed in vitro and ex vivo assays to investigate their inhibitory effects on matrix metalloproteinases (MMPs). As results, LMW feather keratins marginally inhibited collagenase, elastase, and radical scavenging activities. On the other hand, LMW feather keratins significantly suppressed the expression of ultraviolet B (UVB)-induced MMP-1 and MMP-13 in human dermal fibroblasts. Furthermore, phospho-kinase antibody array revealed that LMW feather keratins suppressed UVB-induced phosphorylation of Akts, c-Jun N-terminal kinases 1, p38 beta, and RSK2, but not ERKs in human dermal fibroblast. Overall, these results suggest that LMW feather keratins are potential candidates as cosmeceutical peptides for anti-skin aging.

Obligate sugar oxidation in Mesotoga spp., phylum Thermotogae, in the presence of either elemental sulfur or hydrogenotrophic sulfate-reducers as electron acceptor.

Mesotoga prima strain PhosAc3 is a mesophilic representative of the phylum Thermotogae comprising only fermentative bacteria so far. We show that while unable to ferment glucose, this bacterium is able to couple its oxidation to reduction of elemental sulfur. We demonstrate furthermore that M. prima strain PhosAc3 as well as M. prima strain MesG1 and Mesotoga infera are able to grow in syntrophic association with sulfate-reducing bacteria (SRB) acting as hydrogen scavengers through interspecies hydrogen transfer. Hydrogen production was higher in M. prima strain PhosAc3 cells co-cultured with SRB than in cells cultured alone in the presence of elemental sulfur. We propose that the efficient sugar-oxidizing metabolism by M. prima strain PhosAc3 in syntrophic association with a hydrogenotrophic sulfate-reducing bacterium can be extrapolated to all members of the Mesotoga genus. Genome comparison of Thermotogae members suggests that the metabolic difference between Mesotoga and Thermotoga species (sugar oxidation versus fermentation) is mainly due to the absence of the bifurcating [FeFe]-hydrogenase in the former. Such an obligate oxidative process for using sugars, unusual within prokaryotes, is the first reported within the Thermotogae. It is hypothesized to be of primary ecological importance for growth of Mesotoga spp. in the environments that they inhabit.

Isolation, characterization, and survival strategies of Thermotoga sp. strain PD524, a hyperthermophile from a hot spring in Northern Thailand.

A hyperthermophilic Thermotoga sp. strain PD524 was isolated from a hot spring in Northern Thailand. Cells were long-curved rods (0.5-0.6 × 2.5-10 µm) surrounded by a typical outer membrane toga. Strain PD524 is aero-tolerant at 4 °C but is aero-sensitive at 80 °C. A heat resistant subpopulation was observed in late-stationary phase. Cells from late-stationary phase were revealed remarkably less sensitive to 0.001 % SDS treatment than cells from exponential phase. The temperature range for growth was 70-85 °C (opt. temp. 80 °C), pH range was 6-8.5 (opt. pH 7.5-8.0), and NaCl range of 0 to <10 g/L (opt. 0.5 g/L). Glucose, sucrose, maltose, fructose, xylose, mannose, arabinose, trehalose, starch, and cellobiose were utilized as growth substrates. Growth was inhibited by S(o). Growth yield was stimulated by SO 4 (=) but not by S2O 3 (=) and NO3 (-). Analysis of 16S rRNA gene sequence (KF164213) of strain PD524 revealed closest similarity (96 %) to Thermotoga maritima MSB8(T), T. neapolitana NES(T), T. petrophila RKU-1(T), and T. naphthophila RKU-10(T).

Reconstruction of ancestral 16S rRNA reveals mutation bias in the evolution of optimal growth temperature in the Thermotogae phylum.

Optimal growth temperature is a complex trait involving many cellular components, and its physiology is not yet fully understood. Evolution of continuous characters, such as optimal growth temperature, is often modeled as a one-dimensional random walk, but such a model may be an oversimplification given the complex processes underlying the evolution of continuous characters. Recent articles have used ancestral sequence reconstruction to infer the optimal growth temperature of ancient organisms from the guanine and cytosine content of the stem regions of ribosomal RNA, allowing inferences about the evolution of optimal growth temperature. Here, we investigate the optimal growth temperature of the bacterial phylum Thermotogae. Ancestral sequence reconstruction using a nonhomogeneous model was used to reconstruct the stem guanine and cytosine content of 16S rRNA sequences. We compare this sequence reconstruction method with other ancestral character reconstruction methods, and show that sequence reconstruction generates smaller confidence intervals and different ancestral values than other reconstruction methods. Unbiased random walk simulation indicates that the lower temperature members of the Thermotogales have been under directional selection; however, when a simulation is performed that takes possible mutations into account, it is the high temperature lineages that are, in fact, under directional selection. We find that the evolution of Thermotogales optimal growth temperatures is best fit by a biased random walk model. These findings suggest that it may be easier to evolve from a high optimal growth temperature to a lower one than vice versa.

Bacterial colonization of oral implants from nondental sources.

Implants showing signs of peri-implantitis harbor a microbiota similar to that of periodontitis-affected teeth. This case report describes the subgingival microbiota of a 45-year-old female with advanced periodontitis before and after complete edentulation and reconstruction with dental implants. A 3-month healing period post extraction passed before implants were placed using a two-stage submerged implant protocol. At 4- to 6-month recall visits after definitive prosthetic reconstruction, some implant sites showed bleeding on probing and localized mucositis. Microbiological culture of three inflamed peri-implant sites showed an almost identical spectrum of pathogens, including Porphyromonas gingivalis, Tannerella forsythia, and other major pathogenic bacteria characteristic of aggressive periodontitis. As natural teeth were absent for 8 months, this case report suggests that periodontal pathogens can be retained for a prolonged period of time in nondental sites, from where they can later colonize and compromise the health of dental implants. The therapeutic implications of this finding are discussed.

An in vitro biofilm model of subgingival plaque.

INTRODUCTION: Numerous biofilm models have been described for the study of bacteria associated with the supragingival plaque. However, there are fewer models available for the study of subgingival plaque. The purpose of this study was to develop and validate a model that closely mimicked the composition of the subgingival flora. METHODS: The model was developed as follows: calcium hydroxyapatite disks were coated overnight with 10% sterile saliva, placed in flat-bottomed tissue culture plates containing trypticase-soy broth, directly inoculated with a small aliquot of dispersed subgingival plaque, incubated anaerobically, and transferred to fresh medium at 48-h intervals until climax (steady-state) biofilms were formed ( approximately 10 days). RESULTS: The model, based on samples from eight periodontitis patients and eight healthy subjects, yielded a multi-species, heterogeneous biofilm, consisting of both gram-positive and gram-negative species, and comprising 15-20 cultivable species associated with the subgingival flora. The species present and their proportions were reflective of the initial cultivable subgingival flora. Comparisons of the initial plaque samples from healthy subjects and the mature biofilms showed 81% similarity in species and 70% similarity in the proportions present. Biofilms formed from samples obtained from periodontally diseased subjects were 69% similar in species and 57% similar in the proportions present. CONCLUSIONS: The biofilm model described here closely reproduces the composition of the cultivable subgingival plaque both in the species present and in their relative proportions. Differences existed between biofilms grown from diseased and non-diseased sites with the former being characterized by the presence of periodontal pathogens at microbially significant levels.

Methanol utilizing Desulfotomaculum species utilizes hydrogen in a methanol-fed sulfate-reducing bioreactor.

A sulfate-reducing bacterium, strain WW1, was isolated from a thermophilic bioreactor operated at 65 degrees C with methanol as sole energy source in the presence of sulfate. Growth of strain WW1 on methanol or acetate was inhibited at a sulfide concentration of 200 mg l(-1), while on H2/CO2, no apparent inhibition occurred up to a concentration of 500 mg l(-1). When strain WW1 was co-cultured under the same conditions with the methanol-utilizing, non-sulfate-reducing bacteria, Thermotoga lettingae and Moorella mulderi, both originating from the same bioreactor, growth and sulfide formation were observed up to 430 mg l(-1). These results indicated that in the co-cultures, a major part of the electron flow was directed from methanol via H2/CO2 to the reduction of sulfate to sulfide. Besides methanol, acetate, and hydrogen, strain WW1 was also able to use formate, malate, fumarate, propionate, succinate, butyrate, ethanol, propanol, butanol, isobutanol, with concomitant reduction of sulfate to sulfide. In the absence of sulfate, strain WW1 grew only on pyruvate and lactate. On the basis of 16S rRNA analysis, strain WW1 was most closely related to Desulfotomaculum thermocisternum and Desulfotomaculum australicum. However, physiological properties of strain WW1 differed in some aspects from those of the two related bacteria.

H(2) production and carbon utilization by Thermotoga neapolitana under anaerobic and microaerobic growth conditions.

H(2) production by Petrotoga miotherma, Thermosipho africanus, Thermotoga elfii, Fervidobacterium pennavorans, and Thermotoga neapolitana was compared under microaerobic conditions. Contrary to these previously reported strains being strict anaerobes, all tested strains grew and produced H(2) in the presence of micromolar levels of O(2). T. neapolitana showed the highest H(2) production under these conditions. Microscopic counting techniques were used to determine growth curves and doubling times, which were subsequently correlated with optical density measurements. The Biolog anaerobic microtiter plate system was used to analyze the carbon source utilization spectrum of T. neapolitana and to select non-metabolized or poorly metabolized carbohydrates as physiological buffers. Itaconic acid was successfully used as a buffer to overcome pH-induced limitations of cell growth and to facilitate enhanced production of CO-free H(2).

Marinitoga piezophila sp. nov., a rod-shaped, thermo-piezophilic bacterium isolated under high hydrostatic pressure from a deep-sea hydrothermal vent.

A thermophilic, anaerobic, piezophilic, chemo-organotrophic sulfur-reducing bacterium, designated as KA3T, was isolated from a deep-sea hydrothermal chimney sample collected at a depth of 2630 m on the East-Pacific Rise (13 degrees N). When grown under elevated hydrostatic pressure, the cells are rod-shaped with a sheath-like outer structure, motile, have a mean length of 1-1.5 microm and stain Gram-negative. They appear singly or in short chains. When grown at lower, or atmospheric, pressures, the cells elongate and become twisted. Growth is enhanced by hydrostatic pressure; the optimal pressure for growth is 40 MPa (26 MPa pressure at sampling site). The temperature range for growth is 45-70 degrees C, the optimum being around 65 degrees C (doubling time is approximately 20 min at 40 MPa). Growth is observed from pH 5 to pH 8, the optimum being at pH 6. The salinity range for growth is 10-50 g NaCl l(-1), the optimum being at 30 g l(-1). The isolate is able to grow on a broad spectrum of carbohydrates or complex proteinaceous substrates, and growth is stimulated by L-cystine and elemental sulfur. The G+C content of the genomic DNA is 29 +/- 1 mol%. According to phylogenetic analysis of the 16S rDNA gene, the strain is placed within the order Thermotogales, in the bacterial domain. On the basis of 16S rDNA sequence comparisons and morphological, physiological and genotypic characteristics, it is proposed that the isolate be described as a novel species of the genus Marinitoga, with Marinitoga piezophila sp. nov. as the type species. The type strain is KA3T (= DSM 14283T = JCM 11233T).

Growth requirements and fermentation products of Fusobacterium prausnitzii, and a proposal to reclassify it as Faecalibacterium prausnitzii gen. nov., comb. nov.

Two newly isolated strains of obligately anaerobic bacteria from human faeces are shown here to be related to Fusobacterium prausnitzii, which is regarded as one of the most abundant colonizers of the human colon. These strains, along with Fusobacterium prausnitzii ATCC 27768(T) and 27766, are non-motile and produce butyrate, formate and lactate, but not hydrogen as fermentation products. A new finding is that all four strains produce D-lactate, but not L-lactate. The strains have a requirement for acetate in the growth medium and this may account for the previously reported requirement for rumen fluid. The DNA G+C content of the four strains is 47-57 mol%. Together with phylogenetic analysis based on 16S rRNA sequencing, this establishes that Fusobacterium prausnitzii strains are only distantly related to Fusobacterium sensu stricto and are more closely related to members of Clostridium cluster IV (the Clostridium leptum group). It is proposed that a new genus, Faecalibacterium gen. nov. be created; this genus should include Faecalibacterium prausnitzii gen. nov., comb. nov. ATCC 27768(T) (= NCIMB 13872(T)) (formerly Fusobacterium prausnitzii) as the type species together with ATCC 27766 and the newly isolated strains A2-165 and L2-6.

Phenotypic and genetic data supporting reclassification of Butyrivibrio fibrisolvens isolates.

Among 55 Butyrivibrio fibrisolvens strains five ribotypes of B. fibrisolvens were described on the basis of RFLP profiles of 16S rDNA regions obtained with restriction endonuclease HaeIII. In the phylogenetic tree, these ribotypes were located in the XIVa cluster of Gram-positive bacteria. Phenotypic differences of selected ribotype groups became the basis for further reclassification of B. fibrisolvens.

Application of flow cytometry for ecological monitoring of the rumen microbial ecosystem.

Flow cytometry in combination with fluorescently labeled ribosomal RNA oligonucleotide probes was used for enumeration and monitoring of ruminal bacteria. The polyanionic azo dye Trypan Blue was used for discrimination between live bacterial cells and inorganic particles and the separation was further improved by lysozyme treatment and sonication. Cy3-labeled universally conserved probe EUB338 and FITC-labeled Prevotella bryantii specific probe PBB14 were used for in situ hybridization in mixed culture experiments and in samples of crude rumen fluid. The results were analyzed by flow cytometry. The separation of P. bryantii and Butyrivibrio fibrisolvens, another ruminal bacterium, in mixed culture experiments was satisfactory and enabled monitoring of these bacteria in a test system. P. bryantii cells were detected in crude rumen fluid samples only after supplementation with pure culture cells; this implicates a low concentration of P. bryantii cells in vivo (less than 100/nL, i.e. 10(5) per mL).

Development of competitive PCR for detection of Butyrivibrio fibrisolvens in the rumen.

Competitive PCR method was developed for the detection and enumeration of Butyrivibrio fibrisolvens. Sequences of 16S rDNA were obtained from our isolates (serving as a source of data for primer design) and were distinguished into nine different groups of butyrivibria. Specific primers for two distinct groups were designed with the help of BioEdit program. These primers were tested with DNA of 20 strains of ruminal B. fibrisolvens isolates. Annealing temperature 58 degrees C showed a little specificity but a better selectivity was found after raising it up to 65 degrees C. A group 1 competitive fragment of 16S rDNA of different length was constructed using restriction cutting with MspI followed by ligation; the size of the resulting fragment was cut down by 75 bp. The fragment worked in the presence of the original 16S rDNA fragment of B. fibrisolvens JK 609.

Multiple endoxylanases of Butyrivibrio sp.

Butyrivibrio sp. Mz 5 with a high xylanolytic activity was isolated. Four major xylanases were detected in the cell-associated fraction using the zymogram technique. The xylanolytic activity was inducible with the oat spelts xylan; two endoxylanases (51 and 145 kDa) were formed constitutively. The bulk of the xylanolytic activity was cell-bound and growth-phase dependent; the maximum activity in the cell-associated fraction was achieved after 16 h of incubation. The highest xylanolytic activity was determined in a medium with 0.5% oat spelts xylan. Under optimum conditions (the highest xylanolytic activity produced), the two cell-bound xylanases (51 and 58 kDa) were isolated by anion exchange chromatography on CIM DEAE 8 tubes attached to a MPLC system, and gel filtration.

Distribution and physiological characteristics of hyperthermophiles in the Kubiki oil reservoir in Niigata, Japan.

The distribution of culturable hyperthermophiles was studied in relation to environmental conditions in the Kubiki oil reservoir in Japan, where the temperature was between 50 and 58 degrees C. Dominant hyperthermophilic cocci and rods were isolated and shown to belong to the genera Thermococcus and Thermotoga, respectively, by 16S rDNA analyses. Using the most-probable-number method, we found that hyperthermophilic cocci were widely distributed in several unconnected fault blocks in the Kubiki oil reservoir. In 1996 to 1997, their populations in the production waters from oil wells were 9.2 x 10(3) to 4.6 x 10(4) cells/ml, or 10 to 42% of total cocci. On the other hand, hyperthermophilic rods were found in only one fault block of the reservoir with populations less than 10 cells/ml. Dominant Thermococcus and Thermotoga spp. grew at reservoir temperatures and utilized amino acids and sugars, respectively, as sole carbon sources. While organic carbon was plentiful in the environment, these hyperthermophiles were unable to grow in the formation water due to lack of essential nutrients. Concentrations of some organic and inorganic substances differed among fault blocks, indicating that the movement of formation water between fault blocks was restricted. This finding suggests that the supply of nutrients via fluid current is limited in this subterranean environment and that the organisms are starved in the oil reservoir. Under starved conditions at 50 degrees C, culturable cells of Thermococcus sp. remained around the initial cell density for about 200 days, while those of Thermotoga sp. decreased exponentially to 0. 01% of the initial cell density after incubation for the same period. The difference in survivability between these two hyperthermophiles seems to reflect their populations in the fault blocks. These results indicate that hyperthermophilic cocci and rods adapt to the subterranean environment of the Kubiki oil reservoir by developing an ability to survive under starved conditions.

Chlorobium ferrooxidans sp. nov., a phototrophic green sulfur bacterium that oxidizes ferrous iron in coculture with a "Geospirillum" sp. strain.

A green phototrophic bacterium was enriched with ferrous iron as sole electron donor and was isolated in defined coculture with a spirilloid chemoheterotrophic bacterium. The coculture oxidized ferrous iron to ferric iron with stoichiometric formation of cell mass from carbon dioxide. Sulfide, thiosulfate, or elemental sulfur was not used as electron donor in the light. Hydrogen or acetate in the presence of ferrous iron increased the cell yield of the phototrophic partner, and hydrogen could also be used as sole electron source. Complexed ferric iron was slowly reduced to ferrous iron in the dark, with hydrogen as electron source. Similar to Chlorobium limicola, the phototrophic bacterium contained bacteriochlorophyll c and chlorobactene as photosynthetic pigments, and also resembled representatives of this species morphologically. On the basis of 16S rRNA sequence comparisons, this organism clusters with Chlorobium, Prosthecochloris, and Pelodictyon species within the green sulfur bacteria phylum. Since the phototrophic partner in the coculture KoFox is only moderately related to the other members of the cluster, it is proposed as a new species, Chlorobium ferrooxidans. The chemoheterotrophic partner bacterium, strain KoFum, was isolated in pure culture with fumarate as sole substrate. The strain was identified as a member of the epsilon-subclass of the Proteobacteria closely related to "Geospirillum arsenophilum" on the basis of physiological properties and 16S rRNA sequence comparison. The "Geospirillum" strain was present in the coculture only in low numbers. It fermented fumarate, aspartate, malate, or pyruvate to acetate, succinate, and carbon dioxide, and could reduce nitrate to dinitrogen gas. It was not involved in ferrous iron oxidation but possibly provided a thus far unidentified growth factor to the phototrophic partner.

Identification and characterization of the natural electron donor ferredoxin and of FAD as a possible prosthetic group of benzoyl-CoA reductase (dearomatizing), a key enzyme of anaerobic aromatic metabolism.

Under anoxic conditions most aromatic compounds are metabolized via benzoyl-CoA which becomes reduced by benzoyl-CoA reductase (dearomatizing); this enzyme was recently described in the bacterium Thauera aromatica [Boll, M. & Fuchs, G. (1995) Eur. J. Biochem. 234, 921-933]. It catalyzes the reaction benzoyl-CoA + 2 e- + 2 H+ + 2 MgATP + 2 H2O --> cyclohexa-1,5-diene-1-carboxyl-CoA + 2 MgADP + 2 Pi. The iron-sulfur protein has a native molecular mass of 160-170 kDa and consists of four different subunits. In addition a flavin may be present. The nature of the potential prosthetic group and the natural electron donor were determined. Purified benzoyl-CoA reductase preparations contained 0.25-0.3 mol FAD/mol enzyme. Cells grown anaerobically with aromatic substrates contained a ferredoxin which represented the main, if not the only ferredoxin present. It was purified from 200 g cells with a yield of 60 mg and its N-terminal amino acid sequence was determined. The native molecular mass was 9659 +/- 2 Da as determined by electrospray mass spectrometry. The protein contained 7.6 +/- 0.6 mol iron and 7.6 +/- 1 mol acid-labile sulfur/mol. The ultraviolet-visible spectrum of the protein was typical for ferredoxins with maxima at 280 nm and 390 nm (in the oxidized state). The estimated molar absorption coefficients were 63500 M(-1) cm(-1) at 280 nm and 40500 M(-1) cm(-1) at 390 nm. The difference spectrum between the oxidized and the reduced form had a maximum at 415 nm with delta epsilon415 = 8200 M(-1) cm(-1). 1 mol ferredoxin became reduced/mol dithionite added, suggesting the presence of two [4Fe-4S] clusters. The average midpoint potential of the iron-sulfur clusters was -450 mV. The ferredoxin gene was cloned and sequenced. It was located in a gene cluster coding for enzymes involved in anaerobic aromatic metabolism. The amino acid sequence of the T. aromatica ferredoxin showed high similarities to several other ferredoxins containing 2[4Fe-4S] clusters, e.g. from Clostridia and phototrophic bacteria. The reduced ferredoxin served as electron donor for benzoyl-CoA reduction at a three times higher rate compared with the rate obtained with the artificial electron donor reduced methyl viologen. The turnover number with the natural electron donor of 5 s(-1) can explain the bacterial growth rate with benzoate as substrate. Half-maximal enzyme activity was obtained with 6 microM reduced ferredoxin, at an estimated cellular concentration of 70 microM ferredoxin. Both the low apparent Km value and the turnover number are consistent with the proposed role of ferredoxin in aromatic-ring reduction.