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Keywords:

  • feather degradation;
  • keratinase;
  • anaerobe;
  • KD-1

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

A novel thermophilic, anaerobic, keratinolytic bacterium designated KD-1 was isolated from grassy marshland. Strain KD-1 was a spore-forming rod with a Gram-positive type cell wall, but stained Gram-negative. The temperature, pH, and NaCl concentration range necessary for growth was 30–65 °C (optimum 55 °C), 6.0–10.5 (optimum 8.0–8.5), and 0–6% (optimum 0.2%) (w/v), respectively. Strain KD-1 possessed extracellular keratinase, and the optimum activity of the crude enzyme was pH 8.5 and 70 °C. The enzyme was identified as a thermostable serine-type protease. The strain was sensitive to rifampin, chloramphenicol, kanamycin, and tetracycline and was resistant to erythromycin, neomycin, penicillin, and streptomycin. The main cellular fatty acid was predominantly C15:0 iso (64%), and the G+C content was 28 mol%. Morphological and physiological characterization, together with phylogenetic analysis based on 16S rRNA gene sequencing identified KD-1 as a new species of a novel genus of Clostridiaceae with 95.3%, 93.8% 16S rRNA gene sequence similarity to Clostridium ultunense BST (DSM 10521T) and Tepidimicrobium xylanilyticum PML14T (= JCM 15035T), respectively. We propose the name Keratinibaculum paraultunense gen. nov., sp. nov., with KD-1 (=JCM 18769T =DSM 26752T) as the type strain.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Keratins are insoluble structural proteins that are extensively cross-linked by disulfide bonds, hydrogen bonds, and hydrophobic interactions. Keratins are abundant in hair, feathers, and horns. The α-helix and β-sheet configurations of keratins are characteristically resistant to common proteases including trypsin, pepsin, and papain (Thys et al., 2004; Riffel et al., 2007). However, keratins can be degraded by keratinase secreted by keratinolytic microorganisms (Lin et al., 1992; Boeckle et al., 1995; Bernal et al., 2006; Ionata et al., 2008). Reflecting the ubiquity of keratinolytic microorganisms in natural environments, keratin accumulation does not naturally occur (Williams et al., 1990; Riffel et al., 2007). Keratinolytic microorganisms include bacteria (mainly actinobacteria and bacteria in the genus Bacillus), fungi, and archaea (Brandelli et al., 2010; Korniłłowicz-Kowalska & Bohacz, 2011).

We isolated and characterized a thermophilic, anaerobic, feather-degrading bacterium secreting extracellular serine keratinase. Based on the phenotypic, genotypic, and physiological evidence, strain KD-1 is herein identified as a new species of a novel genus of Clostridiaceae and is designated K. paraultunense KD-1.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Enrichment and isolation

Strain KD-1 was isolated from grassy marshland. The soil was added to an enrichment medium (EM) (L−1: feather 10 g, peptone 10 g, MgSO4·7H2O 0.2 g, L-Cys-HCl 1 g, resazurin 1 mg) in anaerobic bottles sealed with butyl rubber stoppers under a gaseous atmosphere of 100% N2 (Hungate, 1969; Bryant, 1972; Hungate & Macy, 1973). A selective medium (SM) [L−1: feather 10 g,K2HPO4 1 g, KH2PO4 0.4 g, NaCl 3 g, MgSO4·7H2O 0.2 g, vitamin solution 10 mL (DSM 141 medium), trace element solution 10 mL (DSM 141 medium), L-Cys-HCl 1 g L−1, and resazurin 1 mg] was used to isolate feather-degrading bacteria. The medium pH was adjusted to 7.0–7.5 with 5 M KOH. The solid version of the SM was of the same composition, with the exceptions of feather meal as replacement for whole feather and inclusion of 2% (w/v) agar. All media were sterilized by autoclaving at 121 °C for 30 min. Feather-degrading bacteria were isolated by serial dilution and the Hungate roll-tube technique (Hungate, 1969). Enrichment culture (0.4 mL) was serially diluted in 4 mL of solid medium in roll tubes using syringes and needles, and incubated at 55 °C. Single colonies were picked and transferred to the SM medium and incubated at 55 °C. Cell morphology was checked using a model 80i phase-contrast microscope (Nikon, Japan). The roll-tube procedure was repeated several times until a pure culture was obtained.

Morphology

Cell morphology were examined using scanning electron microscopy (SEM) with a JSM-7500F microscope (JEOL, Japan) (Cheng et al., 2008) and by transmission electron microscopy (TEM) using a H-600IV microscope (Hitachi, Japan) (Mikucki et al., 2003). SEM was also used to observe the surface of chicken feathers. Gram stain reaction was determined by a traditional method (Boone & Whitman, 1988) and KOH technique (Buck, 1982). Spore staining was performed conventionally.

G+C content

Genomic DNA was extracted and purified using a TIANamp bacteria DNA extraction kit (TIANGEN Biotech, China). The G+C content was determined by the thermal denaturation method (Marmur & Doty, 1962; Cheng et al., 2007) using a Lambda35 UV/VIS Spectrometer (Perkin/Elmer) with Escherichia coli K12 (CGMCC 1.365) as the reference bacterial strain.

16S rRNA gene sequencing and analysis

The 16S rRNA gene was amplified from the extracted genomic DNA using a PCR kit (TakaraBio, Japan) with primers 27f (5′AGAGTTTGATCMTGGCTCAG) and 1492r (5′TACGGYTACCTTGTTACGACTT) (Karita et al., 2003). The PCR conditions were 94 °C for 5 min, 30 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 2 min; and 72 °C for 10 min. The PCR products were purified using a PCR purification kit (TIAGEN Biotech). Sequence data were performed by GeneWiz. The 16S rRNA gene sequence of strain KD-1 was submitted to GenBank to search for similar sequences using the blast algorithm. The best matching sequences were retrieved from the database and aligned. The similarity analysis was performed using the ClustalW program within MEGA4 (Tamura et al., 2007). Phylogenetic trees were constructed using the neighbor-joining, maximum-likelihood, and maximum-parsimony methods implemented in MEGA4 (Tamura et al., 2007). The phylogenetic tree was evaluated by bootstrap analysis based on 1000 replications.

Cellular fatty acid content

Fatty acid analyses were carried out by the identification service of the DSMZ, Braunschweig, Germany using the Sherlock MIS (MIDI Inc., Newark) system.

Respiratory quinones

Respiratory quinones of strain KD-1T were extracted, separated, and identified as Minnikin et al. (1984) and analyzed by high-performance liquid chromatography (HPLC) (Collins & Jones, 1980).

Physiology and biochemistry analyses

Potential substrate utilization studies were performed in basal medium (SM culture without feather) containing various substrates. The substrates were proteins and sugars at the final concentration of 1% (w/v) and amino acids at a final concentration of 20 mM. The pH was adjusted to 8.0 with 3% (w/v) Na2S and was incubated at 55 °C.

To investigate the utilization of electron acceptors, autoclaved anaerobic 9,10-anthraquinone-2,6-disulfonate (AQDS), FeCl3, Fe2O3, citric acid iron; Na2S2O3, NaNO3, disodium fumarate, Na2SO4, NaNO2, and sodium selenite (final concentration 20 mM) were injected to the SM before the addition of peptone as electron donor (final concentration 1% w/v). Cultivating was carried out at 55 °C. Growth was determined by measuring the optical density at 600 nm (OD600 nm) of cultures, and the products were determined as previously described (Slobodkin et al., 1999; Zavarzina et al., 2002).

Volatile fatty acids (VFA) were analyzed by gas chromatography using an Agilent 7820A system equipped with a DB-FFAP column (30 m × 250 μm×0.5 μm) and a flame ionization detector. Nitrogen was supplied as the carrier gas. The flow rate of nitrogen, hydrogen, and air was 44, 40, and 400 mL min−1, respectively. The injection port and detector temperature were 250 °C. The oven was sequentially maintained at 40 °C for 3 min, 100 °C for 10 min, 180 °C for 5 min, and 200 °C for 15 min. After acidification with 5 M HCl and centrifugation at 12 000 g for 10 min, 1 μL of sample was injected for gas chromatography analysis. A mixture of methanol, ethanol, isobutanol, acetic acid, propionic acid, butyric acid, isobutyric acid, isovaleric acid, and hexanoic acid was used as an external standard.

The growth curve, temperature, pH, and NaCl range for growth were determined by monitoring the OD600 nm of the SM culture. The growth curve test was conducted using optimum conditions of temperature and pH. The effect of temperature was explored using a temperature range of 15–75 °C in 5 °C intervals. The pH range was from 5.0 to 11.0 and was adjusted with HCl or NaOH (1 M). NaCl values ranged from 0 to 10% (w/v).

The antimicrobial susceptibility was tested in SM culture with various antibiotics including neomycin sulfate, ampicillin sodium, streptomycin sulfate, kanamycin sulfate, chloramphenicol, erythromycin, rifampicin, and tetracyline–HCl at a final concentration of 100 μg mL−1. Growth was determined by measuring the OD600 nm of cultures.

All experiments were carried out in triplicate.

Enzyme assays

Keratinase

Keratinase activity was measured using feather meal as the substrate as described previously (Ramnani et al., 2005) with some modifications. Briefly, 2 mL of 50 mM Tris-HCl buffer (pH 8.5) was mixed with 10 mg of feather meal, 1.0 mL of sterile supernatant was added, and the mixture was incubated at 70 °C for 1 h. The reaction was stopped by the addition of 2 mL of 10% trichloroacetic acid (TCA), and the mixture was stored on ice for 10 min prior to centrifugation at 11 963 g for 10 min. The absorbance of the supernatant was measured at 280 nm in a model DU730 spectrophotometer (Beckman). The control was prepared by adding TCA to the reaction mixture before adding the enzyme solution. One unit of keratinase activity was defined as the amount of enzyme that produced an increased absorbance of 0.01.

Thiol group and disulfide reductase

Free thiol content in KD-1 cultures was determined as described previously (Ellman, 1959) with only minor modification (use of reduced glutathione as the standard). A 50 mM working solution of 5,5-dithiobis, 2-nitrobenzoic acid (DTNB) was prepared in a phosphate buffer (pH 8.0) in a 1 : 50 ratio. Two hundred microliters of the working solution was mixed with 20 μL extracellular broth. Absorbance was measured at 412 nm after 15 min at room temperature. Phosphate buffer was used to replace the sample in the control preparations. Disulfide reductase activity was measured as previously described (Ramnani et al., 2005). Extracellular, lysed cell, and lysed broth were tested. One milliliter of enzyme mixture (sample, phosphate buffer (100 mM, pH 8.0), and distilled water in a 3 : 2 : 5 ratio) was incubated with 1 mL of 2 mM oxidized glutathione (dissolved in 100 mM, pH 7.0 phosphate buffer) for 30 min at 55 °C with 5 mM phenylmethanesulfonyl fluoride. The mixture was centrifuged at 1000 g for 10 min, and the supernatant was analyzed for the content of the thiol group. Phosphate buffer (100 mM, pH 7.0) was used instead of oxidized glutathione as the control.

Keratinolytic protease

Crude enzyme was used to study the characteristics of keratinolytic protease. The pH optimum was determined in the pH range 7.0–9.0 with 50 mM Tris-HCl buffer. To determine the optimal temperature for keratinolysis, enzyme reactions were carried out at different temperatures from 45 to 80 °C in Tris-HCl buffer (50 mM, pH 8.5) for 1 h. To investigate thermostability, the crude enzyme was preincubated for 0–210 min at 70, 80, and 90 °C. The residual activity was measured as described above. The effects of metal ions, enzyme inhibitors, detergents, and organic solvents on keratinase activity were ascertained as described above.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Strain KD-1 was a Gram-negative, spore-forming anaerobe. Colonies were white, round, and smooth. Cells were rod shaped (0.3–0.5 × 1.5–2.7 μm) and were arranged alone or linked together end-to-end (Fig. 1a). TEM of ultrathin sections showed a Gram-positive cell wall structure (Fig. 1b). Growth was observed at 30–65 °C (optimum 55 °C), pH 6.0–10.5 (optimum 8.0–8.5), and NaCl concentration of 0–6% (optimum 0.2%). The mean generation time of strain KD-1 was 3.02 h at optimum condition. The strain was sensitive to rifampin, chloramphenicol, tetracycline, and kanamycin and was resistant to erythromycin, neomycin, penicillin, and streptomycin.

image

Figure 1. Cellular morphology of strain KD-1. (a) Representative scanning electron micrograph. The bar denoted 1 μm. (b) Representative transmission electron microscope. The cell membrane (CM) surrounded by a thin, densely peptidoglycan (P), in turn it is surrounded by outer wall (OW).The bar denotes 200 nm.

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Strain KD-1 could degrade native feather within 24 h, and the cells adhered to the surface of feathers (Fig. 2). These findings were similar to those for Bacillus subtilis SLC (Cedrola et al., 2012), B. subtilis S8 (Jeong et al., 2010), and B. licheniformis RG1 (Ramnani et al., 2005). Gelatin, soybean protein, peptone, beef extract, yeast extract, skimmed milk, collagen, casein, chicken feather, duck hair, hair, ox hair, pig hair, and nails could be used as sources of carbon and energy. Strain KD-1 could also use xylan, glycogen, succinate, disodium fumarate, proline + alanine, proline + valine, and proline + serine. Weak growth was observed in the presence of arabinose, maltose, rhamnose, melibiose, raffinose, fructose, sucrose, glucose, xylose, cellobiose, and trehalose. Mannose, amylose, amylopectin, chitin, sodium citrate, sodium hydroxyacetate, sodium carboxymethyl cellulose, 3-hydroxybutyrate, sodium formate, sodium acetate, sodium pyruvate, sodium butyrate, sodium benzoate, sodium malate, 1,2-propylene glycol, methanol, alcohol, 2,3-butanediol, glycerol, olive oil, pectin, D-sorbitol, and betaine were negative. Acetic acid, propionic acid, isobutyric acid, butyric acid, and isovaleric acid were the predominant VFAs produced, with trace amounts of methanol, ethanol, isobutanol, and hexanoic acid produced using peptone or chicken feather as the substrate.

image

Figure 2. Representative scanning electron microscopy images of chicken feathers prior to inoculation with strain KD-1 (a) and 6 h (b), 12 h (c), and 18 h (d) following inoculation with KD-1. After 24 h, the feather morphology was unrecognizable. Arrows indicate cells adherent to the feather surface.

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The electron acceptor test revealed strain KD-1 was able to reduce AQDS, FeCl3, Fe2O3, ferric citrate, but not Na2SO4, NaNO2, and sodium selenite.

The cellular fatty acids of strain KD-1 were C15:0 iso (64.5%), C15:0 iso dma (22.5%), C16:0 dma (2.1%), C16:0 (2.0%), C14:0 dma (1.5%), C11:0 dma (1.5%), C14:0 (1.5%), C17:0 iso dma (1.3%), C17:0 iso (0.7%), C18:0 (0.6%), C18:1ω9c (0.6%), C13:0 iso (0.5%), C18:0 dma (0.5%), and C11:0 iso (0.4%). The G+C content of strain KD-1 was 28 mol% based on Tm. No respiratory quinone was detected in the cells. The near-complete 16S rRNA gene sequence (1491 bp) of strain KD-1 was compared with the most similar sequences retrieved from GenBank. Blast analysis revealed 95.3% and 93.8% similarity with Clostridium ultunense BST and Tepidimicrobium xylanilyticum PML14T, respectively (Fig. 3). Table 1 summarizes some similarities and differences of strain KD-1 with some related taxa. Strain KD-1T had no flagella, while C. ultunense BST and T. xylanilyticum PML14T did. Strain KD-1T produced indole and liquided gelatin and degraded keratin, while C. ultunense BST and T. xylanilyticum PML14T did not. Further, there had almost 20 °C difference between strain KD-1T and C. ultunense BST. Furthermore, C. ultunense BST could oxidize acetate in syntrophic association with hydrogenotrophic methanogenic bacteria, while strain KD-1T did not. The G+C content of strain KD-1, C. ultunense BST and T. xylanilyticum PML14T was 28 mol%, 32 mol%, and 36.2 mol%, respectively, which were significantly different. Therefore, based on the phenotypic, genotypic, and physiological evidence, strain KD-1 was identified as a new species of a novel genus of Clostridiaceae and named K. paraultunense gen. nov., sp. nov.

Table 1. Differential characteristics of strain KD-1 and species most closely related phylogenetically
 1234
  1. Species: 1, Strain KD-1; 2, Clostridium ultunense (DSM 10521T); 3, Tepidimicrobium ferriphilum (DSM 16624T); 4, Tepidimicrobium xylanilyticum (PML14T). ND, Not determined.

Gram staining++++
Spore forming+++
Cell shapeRodRodRodRod
ColoniesWhitish round, 0.5 to 1 mm in diameterWhitish, nearly transparent, disc-shaped, 0.5–1 mm in diameterNDRound, whitish
Cell width ×length (μm)0.3–0.5 × 1.5–2.70.5–0. 7 × 0.5–70.5–0.6 × 3.0–7.00.4–0.5 × 4.0–10.0
Temperature range (optimum) /oC30–65 (55)15–50 (37)26–62 (50)25–67 (60)
pH range (optimum)6.0–10.5 (8.0–8.5)5.0–10.0 (7)5.5–9.5 (7.5–8.0)5.8–9.3 (8.5)
Motility+++
Flagella+++
Indole production++ 
Gelatin hydrolysis+ND
NaCl range (optimum)/(%;w/v)0–6.0% (0.2)0.8M0–3.5%0–4.5% (3.0%)
Keratinolytic+NDNDND
DNA G  +  C content/(mol%)-Tm28323336.2 ± 0.8
Predominant fatty acidsC15:0 iso (64%)NDNDND
Main substrate utilizationSome rigid keratins, peptone, casein, yeast extractFormate, ethylene, glycol, pyruvate, betaine, cysteine, glucosePeptone, tryptone, Casamino acids, yeast extract, beef extract, casein hydrolysate, valine, alanine plus glycine, alanine plusproline, n-propanolNumber of proteinaceous compounds and carbohydrates
ReferenceThis studySchnürer et al. (1996)Slobodkin et al. (2006)Niu et al. (2009)
image

Figure 3. Unrooted phylogenetic tree based on 16S rRNA gene sequences of strain KD-1 and related species using neighbor-joining methods in the program MEGA4. The bar denotes 1% sequence divergence.

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Determination of thiol group and disulfide reductase

Free thiol groups were detected in the extracellular broth. The content of free thiols increased with cultivation time (Supporting information, Fig. S1). No disulfide reductase activity was evident in vitro, suggesting another mechanism of disulfide bond reduction. It is conceivable that KD-1-mediated disulfide bond reduction is important in the adhesion of KD-1 to the feather surface.

Effect of pH and temperature on crude keratinase enzyme activity

The keratinase of strain KD-1 was active at neutral and alkaline pH and exhibited optimum activity at pH 8.5 (Fig. S2a). The enzyme was stable at high temperature (70–90 °C) (Fig. S2b), but keratinase activity declined precipitously at 90 °C. The optimum temperature for keratinase activity of strain KD-1 was 70 °C (Fig. S2c).

Effect of metal ions, enzyme inhibitors, detergents, and organic solvents on keratinase activity

The effects of metal ions, enzyme inhibitors, detergents, and organic solvents at different working concentrations on keratinase activity are summarized in Table 2. This keratinase was strongly inhibited by phenylmethanesulfonyl fluoride and weakly inhibited by EDTA, which confirmed this keratinase as a serine alkaline protease. The reducing agents β-mercaptoethanol and dithiothreitol enhanced enzyme activity by reducing disulfide bonds. Sodium dodecyl sulfate strongly decreased keratinase activity, similar to Bacillus sp. MKR5 (Ghasemi et al., 2012). Zn2+ (20 mM), Ca2+ (5 mM and 20 mM), Mg2+ (5 mM and 20 mM), dimethylsulfoxide, and acetone decreased keratinase activity. Na+, urea, Triton X-100, ethanol, and isopropanol had no significant effects.

Table 2. The effects of metal ion, enzyme inhibitor, detergent, organic solvent, and reducing agent on the crude enzyme activity
FactorsFinal concentration (mM) or [% (v/v)]Activity (%)
  1. Control was defined as 100% activity without influencing factors. SDS, sodium dodecyl sulfate; DMSO, dimethylsulfoxide; DTT, dithiothreitol; PMSF, phenylmethane sulfonylfluoride.

Control 0100.0
Metal ionsNaCl5103.3
2092.7
CaCl2581.0
2076.5
MgCl2581.8
2071.1
ZnSO45104.7
2042.0
Enzyme inhibitorsEDTA170.9
570.5
1046.2
PMSF146.1
518.0
100
DetergentsSDS11.652.5
Urea10095.4
Triton X-1001%105.6
DMSO1%81.3
Organic solventsEthanol3.3%3.3%3.3%109
Isopropanol98.0
Acetone22.5
Reducing agentsβ-mercaptoethanol14.3133.3
DTT1168.9
10290.5

Description of the genus Keratinibaculum

Keratinibaculum [Ke.ra.ti.ni.ba'cu.lum. N.L. n. keratinum, keratin; L. neut. n. baculum, stick, rod; N.L. neut. n. Keratinibaculum, keratin (degrading) rod].

Anaerobic rod-shaped cells with a Gram-positive type cell wall; may stain Gram-negative. Moderately thermophilic and alkaliphilic. The major fatty acids are C15:0 iso, C15:0 iso dma. Low G+C content. Grow on a number of proteinaceous and some saccharides. Proteolytic. The type species is K. parault-unense.

Description of K. paraultunense gen. nov., sp. nov

Keratinibaculum paraultunense [paraultunense: pa.ra.ul.tun.en'se. Gr. prep. para, beside, near; N.L. neut. adj. ultunense, a bacterial-specific epithet; N.L. paraultunense, near (Clostridium) ultunense, phylogenetically related to C. ultunense].

The morphological, chemotaxonomic, and general characteristics are as described for the genus. Cells are 0.3–0.5 × 1.5–2.7 μm, grows singly or in pairs. Spore-forming, Colonies are white, round, and smooth. The temperature, pH, and NaCl range (optimal) for growth is 30–65 °C (55 °C), 6.0–10.5 (8.0–8.5), and 0–6% (0.2%) (w/v), respectively. Utilize proteins and saccharides such as gelatin, soybean protein, peptone, beef extract, yeast extract, skimmed milk, collagen, casein, chicken feather, duck hair, animal hair and nails, xylan, glycogen, succinate, disodium fumarate, proline + alanine, proline + valine, and proline + serine as sources of carbon and energy is observed. Weak growth was observed in the presence of arabinose, maltose, rhamnose, melibiose, raffinose, fructose, sucrose, glucose, xylose, cellobiose, and trehalose. Mannose, amylose, amylopectin, chitin, sodium citrate, sodium hydroxyacetate, sodium carboxymethyl cellulose, 3-hydroxybutyrate, sodium formate, sodium acetate, sodium pyruvate, sodium butyrate, sodium benzoate, sodium malate, 1,2-propylene glycol, methanol, alcohol, 2,3-butanediol, glycerol, olive oil, pectin, D-sorbitol, and betaine are negative. Acetic acid, propionic acid, isobutyric acid, butyric acid, and isovaleric acid are produced on peptone medium. Indole and thiol are produced. AQDS, FeCl3, Fe2O3, and ferric citrate are reduced, Na2SO4, NaNO2, and sodium selenite are not. The cellular fatty acids are C15:0 iso, C15:0 iso dma, C16:0 dma, C16:0, C14:0 dma, C11:0 dma, C14:0, C17:0 iso dma, C17:0 iso, C18:0, C18:1 ω9c, C13:0 iso, C18:0 dma, and C11:0 iso. The G+C content is 28% (Tm). No respiratory quinone was detected. Excretes thermophilic alkaline keratinase, degrades keratin. The type strain is KD-1T (= JCM 18769T = DSM 26752T).

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

We thank Anna Schnürer for supplying the genome of C. ultunense and J.P. Euzéby for the etymology of the novel taxon. This study was supported by the Science Infrastructure Platform of Sichuan province and National High Technology Research and Development Program of China (863 Program) (grants 2013AA102805).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgments
  7. References
  8. Supporting Information
  • Bernal C, Cairo J & Coello N (2006) Purification and characterization of a novel exocellular keratinase from Kocuria rosea. Enzyme Microb Technol 38: 4954.
  • Boeckle B, Galunsky B & Mueller R (1995) Characterization of a keratinolytic serine proteinase from Streptomyces pactum DSM 40530. Appl Environ Microbiol 61: 37053710.
  • Boone DR & Whitman WB (1988) Proposal of minimal standards for describing new taxa of methanogenic bacteria. Int J Syst Bacteriol 38: 212219.
  • Brandelli A, Daroit DJ & Riffel A (2010) Biochemical features of microbial keratinases and their production and applications. Appl Microbiol Biotechnol 85: 17351750.
  • Bryant M (1972) Commentary on the Hungate technique for culture of anaerobic bacteria. Am J Clin Nutr 25: 13241328.
  • Buck JD (1982) Nonstaining (KOH) method for determination of gram reactions of marine bacteria. Appl Environ Microbiol 44: 992993.
  • Cedrola SML, de Melo ACN, Mazotto AM et al. (2012) Keratinases and sulfide from Bacillus subtilis SLC to recycle feather waste. World J Microbiol Biotechnol 28: 12591269.
  • Cheng L, Qiu TL, Yin XB, Wu XL, Hu GQ, Deng Y & Zhang H (2007) Methermicoccus shengliensis gen. nov., sp. nov., a thermophilic, methylotrophic methanogen isolated from oil-production water, and proposal of Methermicoccaceae fam. nov. Int J Syst Evol Microbiol 57: 29642969.
  • Cheng L, Qiu TL, Li X, Wang WD, Deng Y, Yin XB & Zhang H (2008) Isolation and characterization of Methanoculleus receptaculi sp. nov. from Shengli oil field, China. FEMS Microbiol Lett 285: 6571.
  • Collins M & Jones D (1980) Lipids in the classification and identification of coryneform bacteria containing peptidoglycans based on 2,4-diaminobutyric acid. J Appl Microbiol 48: 459470.
  • Ellman GL (1959) Tissue sulfhydryl groups. Arch Biochem Biophys 82: 7077.
  • Ghasemi Y, Shahbazi M, Rasoul-Amini S, Kargar M, Safari A, Kazemi A & Montazeri-Najafabady N (2012) Identification and characterization of feather-degrading bacteria from keratin-rich wastes. Ann Microbiol 62: 737744.
  • Hungate R (1969) A roll-tube method for cultivation of strict anaerobes. Methods Microbiol 3B: 117132.
  • Hungate R & Macy J (1973) The roll-tube method for cultivation of strict anaerobes. Bull Ecol Res Comm 17: 123126.
  • Ionata E, Canganella F, Bianconi G et al. (2008) A novel keratinase from Clostridium sporogenes bv. pennavorans bv. nov., a thermotolerant organism isolated from solfataric muds. Microbiol Res 163: 105112.
  • Jeong JH, Jeon YD, Lee OM, Kim JD, Lee NR, Park GT & Son HJ (2010) Characterization of a multifunctional feather-degrading Bacillus subtilis isolated from forest soil. Biodegradation 21: 10291040.
  • Karita S, Nakayama K, Goto M, Sakka K, Kim WJ & Ogawa S (2003) A novel cellulolytic, anaerobic, and thermophilic bacterium, Moorella sp. strain F21. Biosci Biotech Biochem 67: 183185.
  • Korniłłowicz-Kowalska T & Bohacz J (2011) Biodegradation of keratin waste: theory and practical aspects. Waste Manage 31: 16891701.
  • Lin X, Lee CG, Casale ES & Shih JCH (1992) Purification and characterization of a keratinase from a feather-degrading Bacillus licheniformis strain. Appl Environ Microbiol 58: 32713275.
  • Marmur J & Doty P (1962) Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J Mol Biol 5: 109118.
  • Mikucki JA, Liu Y, Delwiche M, Colwell FS & Boone DR (2003) Isolation of a methanogen from deep marine sediments that contain methane hydrates, and description of Methanoculleus submarinus sp. nov. Appl Environ Microbiol 69: 33113316.
  • Minnikin D, O'Donnell A, Goodfellow M, Alderson G, Athalye M, Schaal A & Parlett J (1984) An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Meth 2: 233241.
  • Niu L, Song L, Liu X & Dong X (2009) Tepidimicrobium xylanilyticum sp. nov., an anaerobic xylanolytic bacterium, and emended description of the genus Tepidimicrobium. Int J Syst Evol Microbiol 59: 26982701.
  • Ramnani P, Singh R & Gupta R (2005) Keratinolytic potential of Bacillus licheniformis RG1: structural and biochemical mechanism of feather degradation. Can J Microbiol 51: 191196.
  • Riffel A, Brandelli A, Bellato CM, Souza GHMF, Eberlin MN & Tavares FCA (2007) Purification and characterization of a keratinolytic metalloprotease from Chryseobacterium sp. kr6. J Biotechnol 128: 693703.
  • Schnürer A, Schink B & Svensson BH (1996) Clostridium ultunense sp. nov., a mesophilic bacterium oxidizing acetate in syntrophic association with a hydrogenotrophic methanogenic bacterium. Int J Syst Bacteriol 46: 11451152.
  • Slobodkin A, Tourova T, Kuznetsov B, Kostrikina N, Chernyh N & Bonch-Osmolovskaya E (1999) Thermoanaerobacter siderophilus sp. nov., a novel dissimilatory Fe (III)-reducing, anaerobic, thermophilic bacterium. Int J Syst Evol Microbiol 49: 14711478.
  • Slobodkin A, Tourova T, Kostrikina N, Lysenko A, German K, Bonch-Osmolovskaya E & Birkeland NK (2006) Tepidimicrobium ferriphilum gen. nov., sp. nov., a novel moderately thermophilic, Fe (III)-reducing bacterium of the order Clostridiales. Int J Syst Evol Microbiol 56: 369372.
  • Tamura K, Dudley J, Nei M & Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24: 15961599.
  • Thys R, Lucas F, Riffel A, Heeb P & Brandelli A (2004) Characterization of a protease of a feather-degrading Microbacterium species. Lett Appl Microbiol 39: 181186.
  • Williams C, Richter C, MacKenzie J Jr & Shih JCH (1990) Isolation, identification, and characterization of a feather-degrading bacterium. Appl Environ Microbiol 56: 15091515.
  • Zavarzina D, Tourova T, Kuznetsov B, Bonch-Osmolovskaya E & Slobodkin A (2002) Thermovenabulum ferriorganovorum gen. nov., sp. nov., a novel thermophilic, anaerobic, endospore-forming bacterium. Int J Syst Evol Microbiol 52: 17371743.

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgments
  7. References
  8. Supporting Information
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fml12184-sup-0001-FigsS1-S2.docxWord document8811K

Fig. S1. Free sulfydryl groups in the extracellular broth measured over time.

Fig. S2. The effect of pH (a) and temperature (b and c) on crude enzyme activity.

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