The sulfur formation system mediating extracellular cysteine-cystine recycling in Fervidobacterium islandicum AW-1 is associated with keratin degradation

Most extremophilic anaerobes possess a sulfur formation (Suf) system for Fe–S cluster biogenesis. In addition to its essential role in redox chemistry and stress responses of Fe–S cluster proteins, the Suf system may play an important role in keratin degradation by Fervidobacterium islandicum AW-1. Comparative genomics of the order Thermotogales revealed that the feather-degrading F. islandicum AW-1 has a complete Suf-like machinery (SufCBDSU) that is highly expressed in cells grown on native feathers in the absence of elemental sulfur (S⁰). On the other hand, F. islandicum AW-1 exhibited a significant retardation in the Suf system-mediated keratin degradation in the presence of S⁰. Detailed differential expression analysis of sulfur assimilation machineries unveiled the mechanism by which an efficient sulfur delivery from persulfurated SufS to SufU is achieved during keratinolysis under sulfur starvation. Indeed, addition of SufS–SufU to cell extracts containing keratinolytic proteases accelerated keratin decomposition in vitro under reducing conditions. Remarkably, mass spectrometric analysis of extracellular and intracellular levels of amino acids suggested that redox homeostasis within cells coupled to extracellular cysteine and cystine recycling might be a prerequisite for keratinolysis. Taken together, these results suggest that the Suf-like machinery including the SufS–SufU complex may contribute to sulfur availability for an extracellular reducing environment as well as intracellular redox homeostasis through cysteine released from keratin hydrolysate under starvation conditions.     

Bacterial community profiles on feathers during composting as determined by terminal restriction fragment length polymorphism analysis of 16S rDNA genes

Composting is one of the more economical and environmentally safe methods of recycling feather waste generated by the poultry industry, since 90% of the feather weight consists of crude keratin protein, and feathers contain 15% N. However, the keratin in waste feathers is resistant to biodegradation and may require the addition of bacterial inocula to enhance the degradation process during composting. Two keratin-degrading bacteria isolated from plumage of wild songbirds and identified as Bacillus licheneformis (OWU 1411T) and Streptomyces sp. (OWU 1441) were inoculated into poultry feather composts (1.13×10⁸ cfu g^–1 feathers) and co-composted with poultry litter and straw in 200-l compost vessels. Composting temperatures, as well as CO₂ and NH₃ evolution, were measured in these vessels to determine the effects of inoculation on the rate and extent of poultry feather decomposition during composting. Terminal restriction fragment length polymorphisms of 16S rRNA genes were used to follow changes in microbial community structure during composting. The results indicated that extensive carbon conversion occurred in both treatments (55.5 and 56.1%). The addition of the bacterial inocula did not enhance the rate of waste feather composting. The microbial community structure over time was very similar in inoculated and uninoculated waste feather composts.     

Degradation of keratin substrates by fungi isolated from sewage sludge

Four fungal species including two dermatophytes and two saprophytes were isolated from sewage sludge samples at Basrah (Iraq) they were tested for their degradative ability towards three types of keratin substrates (human hair, chicken feathers and wool). The rate of keratin degradation was expressed as weight loss over three weeks of incubation using a liquid culture medium. Human hair had the highest degradation rate by colonization of Chrysosporium pannicola and Microsporum gypseum at a rate of 62% and 4% respectively. Chicken feathers were highly degraded by Aspergillus flavus (32%) while wool degradation was highest by C. pannicola (45.5%) and Trichophyton mentagrophytes var. erinacei (38%). There was a significant difference (p < 0.00l) in keratin substrate degradation rates by the examined fungi. Keratinase activity was highest for C. pannicola and M. gypseum in the culture medium baited with human hair. Aspergillus flavus revealed the highest activity of this enzyme in cultures amended with chicken feathers while T. mentagrophytes var. erinacei showed highest keratinase activity in cultures with wool substrate. The amount of protein released into the culture medium varied among the tested fungi. The medium's alkalinity increased over incubation time from 6.5 to 7.8. Microscopic examination showed maceration of the keratin substrates by the fungi. This revised version was published online in June 2006 with corrections to the Cover Date.     

Identification of Genes Preferentially Expressed in Goat Hair Follicle Anagen-Catagen Transition Using Suppression Subtractive Hybridization

Suppressive subtraction hybridization (SSH) was used to identify differentially expressed genes in goat (Capra hircus) hair follicle anagen-catagen transition. The cDNA fragments, derived from SSH positive subtractive library (tester: anagen-catagen transition, driver: later anagen), were cloned into pEGM-T vector. Two hundred cDNA fragments screened from this library were subjected to identify forty-five unregulated isolates. Sequence analysis revealed that these fragments represented twenty-three genes. Blasting analysis with database in GenBank showed that twenty genes were previously clearly annotated, two were homologous to un-annotated expressed sequence tag (ESTs), and one might be novel. To identify characters of gene expression, seven genes in later anagen and anagen-catagen transition skin tissues were chosen for quantitative real-time PCR. Results indicated that expression of these seven genes varied much, reaching threefold among them, furthering indicating that expression of those genes was up-regulation in the anagen-catagen transition. We characterized expression levels of this potential novel gene and the goat ectodysplasin A during differential stages of hair cycle. These profiles suggested that these two genes might play a role in the goat secondary hair follicle cycle.     

Keratinases and sulfide from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> SLC to recycle feather waste

The aim of this study is to investigate the culture conditions of chicken feather degradation and keratinolytic enzyme production by the recently isolated Bacillus subtilis SLC and to evaluate the potential of the SLC strain to recycle feather waste discarded by the poultry industry. The SLC strain was isolated from the agroindustrial waste of a poultry farm in Brazil and was confirmed to belong to Bacillus subtilis by rDNA gene analysis. There was high keratinase production when the medium was at pH 8 (280 U ml⁻¹). Activity was higher using the inoculum propagated for 72 h on 1% whole feathers supplemented with 0.1% yeast extract. In the enzymatic extract, the keratinases were active in the pH range from 2.0 to 12.0 with a maximum activity at pH 10.0 and temperature 60°C. For gelatinase the best pH was 5.0 and the best temperature was 37°C. All keratinases are serine peptidases. The crude enzymatic extract degraded keratin, gelatin, casein, and hemoglobin. Scanning electron microscopy showed Bacillus cells adhered onto feather surfaces after 98 h of culture and degraded feather filaments were observed. MALDI-TOF mass spectrometric analysis showed multiple peaks from 522 to 892 m/z indicating feather degradation. The presence of sulfide was detected on extracellular medium probably participating in the breakdown of sulfide bridges of the feather keratin. External addition of sulfide increased feather degradation.     

Sustainable and practical degradation of intact chicken feathers by cultivating a newly isolated thermophilic <Emphasis Type="Italic">Meiothermus ruber</Emphasis> H328

The sustainable and practical degradation of intact chicken feathers by a newly isolated thermophilic bacterium Meiothermus ruber H328 was presented with extensive data. Aerobic cultivation with moderately thermophilic strain H328 at 55°C for 6 days led to the apparently complete decay of the truly intact feathers and provided 1.89 mmol free amino acids and 7.32 mmol acid-hydrolyzed amino acids from 50 ml of culture containing 3% (w/v) intact chicken feathers. The amino acid components in the soluble fraction of the culture conspicuously agreed with those calculated from the intact feathers. This demonstrated that more than 55% of total keratin proteins were solubilized from the intact chicken feathers into the culture in the forms of free amino acid and/or soluble oligopeptide, and most of them are directly derived from the intact feathers by proteolytic digestion. Feather degradation by strain H328 surpasses that by any other microorganisms with regard to degradation efficiency, absence of requirement for pretreatment of the feathers, and product fidelity in the amino acid component. Furthermore, the culture containing the degradative products from the intact feathers was subjected to matrix-assisted laser desorption ionization mass spectrometry-time-of-flight analysis, and it was revealed that the molecular masses of the solubilized products, oligopeptides, were less than 1,000. This result allows us to investigate the bioactivities of oligopeptides derived from the degradation of chicken feathers by cultivation with strain H328 as well as the production of amino acids for feedstuffs.     

Colourful parrot feathers resist bacterial degradation

The brilliant red, orange and yellow colours of parrot feathers are the product of psittacofulvins, which are synthetic pigments known only from parrots. Recent evidence suggests that some pigments in bird feathers function not just as colour generators, but also preserve plumage integrity by increasing the resistance of feather keratin to bacterial degradation. We exposed a variety of colourful parrot feathers to feather-degrading Bacillus licheniformis and found that feathers with red psittacofulvins degraded at about the same rate as those with melanin and more slowly than white feathers, which lack pigments. Blue feathers, in which colour is based on the microstructural arrangement of keratin, air and melanin granules, and green feathers, which combine structural blue with yellow psittacofulvins, degraded at a rate similar to that of red and black feathers. These differences in resistance to bacterial degradation of differently coloured feathers suggest that colour patterns within the Psittaciformes may have evolved to resist bacterial degradation, in addition to their role in communication and camouflage.     

Isolation and Characterization of an Anther-Specific Polygalacturonase Gene, BcMF16, in Brassica campestris ssp. chinensis

Many genes in the genic male sterile A/B line (Bajh97-01A/B) of Chinese cabbage pak choi (Brassica campestris L. subsp. chinensis Makino) are expressed differentially, and some play critical roles in the formation of pollen walls. In this study, one of these genes, Brassica campestris Male Fertility 16 (BcMF16), has been isolated and characterized. The BcMF16 gene shares approximately 85% nucleotide sequence homology with two exopolygalacturonase (EC3.2.1.67) genes of Arabidopsis thaliana. Cluster analysis of polygalacturonase peptides indicate that BcMF16 belongs to the pollen polygalacturonase clade. Quantitative real-time PCR analysis has revealed that BcMF16 is specifically expressed in reproductive tissues of the fertile line of genic male sterile A/B line of Chinese cabbage pak choi, and that expression levels dramatically increased during later stages of pollen development. In situ hybridization has demonstrated that BcMF16 is specifically and transiently expressed in both tapetum and pollen following microspore separation at the tetrad stage.     

Biphasic and Dually Coordinated Expression of the Genes Encoding Major Shell Matrix Proteins in the Pearl Oyster Pinctada fucata

Regional expression patterns of shell matrix protein genes of Pinctada fucata were investigated using real-time quantitative polymerase chain reaction (PCR) and in situ hybridization. Six shell matrix proteins examined in this study indicated a distinct biphasic pattern of expression, falling into one of the following three groups: (1) expressed only in the more dorsal region of the mantle (MSI60 and N16); (2) expressed only in the more ventral region (MSI31, Prismalin-14, and Aspein); and (3) expressed in both regions (nacrein). The ubiquity of the last protein probably reflects its general role as a carbonate-producing enzyme, while the other groups are interpreted as corresponding to the distinction between the two varieties of shell layers, the aragonitic nacreous layer and the calcitic prismatic layer. In addition, the constituent genes of each of these two groups indicated similar levels of relative expression among different sites even among different individuals, suggesting that the genes of each group share a single upstream regulatory factor, respectively, and that these genes are expressed in a dually coordinated fashion.     

Expression patterns of cotton chloroplast genes during development: implications for development of plastid transformation vectors

Although most plastid transformation studies have focused on chloroplast expression, plastid transformation can also be used to express genes in plastids of a wide variety of plant tissues by using appropriate plastid promoters. Based on the sequence of the Gossypium hirsutum chloroplast genome, we developed primers and amplified segments of 20 different plastid genes. The PCR products were labeled and used in filter dot blot hybridization studies to characterize their expression levels and patterns in total RNA isolated from light- and dark-grown cotton tissues at different developmental stages. A subset of 6 genes among these was further characterized by real time PCR. Highest expression levels were observed for rrn16 and psbA. Four genes were expressed in all samples at relatively constant levels: accD, atpA, matK and rrn16. Expression in root tissue was generally low. The results of our study can be used to predict which operons and promoters are most likely to be preferentially expressed in the plastids of tissues of interest at levels that would result in the desired phenotype, facilitating the development of plastid transformation vectors.     

Genome‐wide screen of genes imprinted in sorghum endosperm,and the roles of allelic differential cytosine methylation

Imprinting is an epigenetic phenomenon referring to allele‐biased expression of certain genes depending on their parent of origin. Accumulated evidence suggests that, while imprinting is a conserved mechanism across kingdoms, the identities of the imprinted genes are largely species‐specific. Using deep RNA sequencing of endosperm 14 days after pollination in sorghum, 5683 genes (29.27% of the total 19 418 expressed genes) were found to harbor diagnostic single nucleotide polymorphisms between two parental lines. The analysis of parent‐of‐origin expression patterns in the endosperm of a pair of reciprocal F₁ hybrids between the two sorghum lines led to identification of 101 genes with ≥ fivefold allelic expression difference in both hybrids, including 85 maternal expressed genes (MEGs) and 16 paternal expressed genes (PEGs). Thirty of these genes were previously identified as imprinted in endosperm of maize (Zea mays), rice (Oryza sativa) or Arabidopsis, while the remaining 71 genes are sorghum‐specific imprinted genes relative to these three plant species. Allele‐biased expression of virtually all of the 14 tested imprinted genes (nine MEGs and five PEGs) was validated by pyrosequencing using independent sources of RNA from various developmental stages and dissected parts of endosperm. Forty‐six imprinted genes (30 MEGs and 16 PEGs) were assayed by quantitative RT–PCR, and the majority of them showed endosperm‐specific or preferential expression relative to embryo and other tissues. DNA methylation analysis of the 5’ upstream region and gene body for seven imprinted genes indicated that, while three of the four PEGs were associated with hypomethylation of maternal alleles, no MEG was associated with allele‐differential methylation.     

Keratinolytic potential of a novel Bacillus sp. P45 isolated from the Amazon basin fish Piaractus mesopotamicus

Bacillus sp. P45, isolated from the intestine of the Amazon basin fish Piaractus mesopotamicus, showed proteolytic activity when grown on skimmed milk and feather meal agar plates. The keratinolytic potential of this strain was evaluated on whole feather broth and human hair broth. Bacillus sp. P45 degraded almost 90% of chicken feathers after 72 h of submerged cultivation on whole feather broth, and the production of extracellular proteases was observed. The formation of thiol groups was also detected during growth, indicating the contribution of sulphitolysis to the efficient hydrolysis of feather keratin. Nevertheless, Bacillus sp. P45 was unable to degrade hair keratin, possibly due to the conformational diversity of this substrate in comparison to feather keratin. Additionally, preliminary results demonstrated that this strain might be utilized in the degradation of recalcitrant collagen-containing wastes. The keratinolytic character of Bacillus sp. P45 might be utilized in environmental-friendly processes such as bioconversion of waste feathers, representing an alternative way of waste management that could lead to the production of value-added products such as microbial biomass, protein hydrolysates and proteolytic enzymes.     

Environmental-induced acquisition of nuptial plumage expression: a role of denaturation of feather carotenoproteins?

Several avian species show a bright carotenoid-based coloration during spring and following a period of duller coloration during the previous winter, despite carotenoids presumably being fully deposited in feathers during the autumn moult. Carotenoid-based breast feathers of male linnets (Carduelis cannabina) increased in hue (redness), saturation and brightness after exposing them to outdoor conditions from winter to spring. This represents the first experimental evidence showing that carotenoid-based plumage coloration may increase towards a colourful expression due to biotic or abiotic environmental factors acting directly on full-grown feathers when carotenoids may be fully functional. Sunlight ultraviolet (UV) irradiation was hypothesized to denature keratin and other proteins that might protect pigments from degradation by this and other environmental factors, suggesting that sunlight UV irradiation is a major factor in the colour increase from winter to spring. Feather proteins and other binding molecules, if existing in the follicles, may be linked to carotenoids since their deposition into feathers to protect colourful features of associated carotenoids during the non-breeding season when its main signalling function may be relaxed. Progress towards uncovering the significance of concealment and subsequent display of colour expression should consider the potential binding and protecting nature of feather proteins associated with carotenoids.     

Biodegradation of feather wastes and the purification and characterization of a concomitant keratinase from Paecilomyces lilacinus

Paecilomyces lilacinus strain PL-HN-16 was found to have the ability to degrade feathers. During the degradation process, the broth initially turned as sticky as gelatin and then turned into fluid that means the feathers can be hydrolyzed completely. Keratinolytic protein (Ker) of aforementioned strain was purified using ammonium sulphate precipitation, HiTrap? Butyl FF chromatography and Sephacryl S-200 gel filtration. The Ker of P. lilacinus PL-HN-16 had molecular mass of 33 kDa, the optimum pH 8.0 and temperature optimum at 40°C. It used the soluble keratin as substrate. The enzyme showed high activity and stability over a wide range of pH (6.0 to 10.0) and temperature (30°C to 60°C) values but was completely inhibited by PMSF. Ker of P. lilacinus PL-HN-16 exhibited stability toward SDS. These promising properties make the enzyme a potential candidate for future applications in biotechnological processes as keratin hydrolysis and dehairing during leather processing.