The gene encoding a cutinase homolog, LC-cutinase, was cloned from a fosmid library of a leaf-branch compost metagenome by functional screening using tributyrin agar plates. LC-cutinase shows the highest amino acid sequence identity of 59.7% to Thermomonospora curvata lipase. It also shows the 57.4% identity to Thermobifida fusca cutinase. When LCcutinase without a putative signal peptide was secreted to the periplasm of Escherichia coli cells with the assistance of the pelB leader sequence, more than 50% of the recombinant protein, termed LC-cutinase*, was excreted into the extracellular medium. It was purified and characterized. LC-cutinase* hydrolyzed various fatty acid monoesters with acyl chain lengths of 2 to 18, with a preference for short-chain substrates (C 4 substrate at most) most optimally at pH 8.5 and 50°C, but could not hydrolyze olive oil. It lost activity with half-lives of 40 min at 70°C and 7 min at 80°C. LC-cutinase* had an ability to degrade poly(-caprolactone) and polyethylene terephthalate (PET). The specific PET-degrading activity of LC-cutinase* was determined to be 12 mg/h/mg of enzyme (2.7 mg/h/kat of pNP-butyrate-degrading activity) at pH 8.0 and 50°C. This activity is higher than those of the bacterial and fungal cutinases reported thus far, suggesting that LC-cutinase* not only serves as a good model for understanding the molecular mechanism of PET-degrading enzyme but also is potentially applicable for surface modification and degradation of PET. C utinase (EC 3.1.1.74) is a lipolytic/esterolytic enzyme that hydrolyzes not only cutin, which is a major component of plant cuticle (38), but also water-soluble esters and insoluble triglycerides (12). It hydrolyzes these substrates to carboxylic acids and alcohols through the formation of an acyl enzyme intermediate, in which the active-site serine residue is acylated by the substrate. This serine residue is located within a GXSXG sequence motif and forms a catalytic triad with His and Asp. Cutinase has been found in both fungi and bacteria. The crystal structures of two fungal cutinases from Fusarium solani f. sp. pisi (22) and Glomerella cingulata (27) have been determined. According to these structures, cutinase shares a common ␣/␤ hydrolase fold with lipase and esterase (28). However, cutinase, like esterase, does not have a lid structure, which is responsible for interfacial activation of lipase (8). Therefore, cutinase does not show interfacial activation like esterase (14). Cutinase has recently received much attention because of its potential application for surface modification and degradation of aliphatic and aromatic polyesters (16), especially polyethylene terephthalate (PET), which is a synthetic aromatic polyester composed of terephthalic acid (TPA) and ethylene glycol (10,16,36,39). However, the number of cutinases, which have been studied regarding PET modification, is still limited, and this limitation may result in the delay of the research toward the practical use of cutinases. Therefore, isolation of a novel cutinase...

Ancient starch research illuminates aspects of human ecology and economic botany that drove human evolution and cultural complexity over time, with a special emphasis on past technology, diet, health, and adaptation to changing environments and socio-economic systems. However, lapses in prevailing starch research demonstrate the exaggerated expectations for the field that have been generated over the last few decades. This includes an absence of explanation for the millennial-scale survivability of a biochemically degradable polymer, and difficulties in establishing authenticity and taxonomic identification. This paper outlines new taphonomic and authenticity criteria to guide future work toward designing research programs that fully exploit the potential of ancient starch while considering growing demands from readers, editors, and reviewers that look for objective compositional identification of putatively ancient starch granules.

Oil sands process-affected water (OSPW), produced by surface-mining of oil sands in Canada, is alkaline and contains high concentrations of salts, metals, naphthenic acids, and polycyclic aromatic compounds (PAHs). Residual hydrocarbon biodegradation occurs naturally, but little is known about the hydrocarbon-degrading microbial communities present in OSPW. In this study, aerobic oxidation of benzene and naphthalene in the surface layer of an oil sands tailings pond were measured. The potential oxidation rates were 4.3 μmol L−1 OSPW d−1 for benzene and 21.4 μmol L−1 OSPW d−1 for naphthalene. To identify benzene and naphthalene-degrading microbial communities, metagenomics was combined with stable isotope probing (SIP), high-throughput sequencing of 16S rRNA gene amplicons, and isolation of microbial strains. SIP using 13C-benzene and 13C-naphthalene detected strains of the genera Methyloversatilis and Zavarzinia as the main benzene degraders, while strains belonging to the family Chromatiaceae and the genus Thauera were the main naphthalene degraders. Metagenomic analysis revealed a diversity of genes encoding oxygenases active against aromatic compounds. Although these genes apparently belonged to many phylogenetically diverse taxa, only a few of these taxa were predominant in the SIP experiments. This suggested that many members of the community are adapted to consuming other aromatic compounds, or are active only under specific conditions. 16S rRNA gene sequence datasets have been submitted to the Sequence Read Archive (SRA) under accession number SRP109130. The Gold Study and Project submission ID number in Joint Genome Institute IMG/M for the metagenome is Gs0047444 and Gp0055765.

An aerobic chemoheterotrophic gliding bacterium, designated RYGT, was isolated from a soil in Germany. Cells were Gram-stain-negative, thin rods (0.4–0.6 µm in width and 2.0–5.5 µm in length). Cells multiplied by normal cell division and no resting stages were observed. Colonies were yellow and displayed swarming edges. Gliding motility was observed in wet mounts. Strain RYGT grew at pH 5.6–7.7 (optimum pH 6.6–7.0), at 13–37 °C (optimum 25–30 °C) and with 0–1.0 % NaCl (optimum 0–0.1 %). The isolate was incapable of atmospheric nitrogen fixation and grew on most mono- and disaccharides as well as a few polysaccharides and organic acids. The predominant menaquinone was MK-7, the major cellular fatty acids were C16 : 1ω5c and iso-C15 : 0 and the major intact polar lipids were composed of phosphatidylethanolamine derivatives and two unknown series. The DNA G+C content was 49.9 mol%. Based on 16S rRNA gene sequence analysis, the isolate belonged to the phylum Bacteroidetes , class Cytophagia , order Cytophagales , but was only distantly related to any cultured bacteria. The closest relatives were Ohtaekwangia koreensis 3B-2T and Ohtaekwangia kribbensis 10AOT (both 93 % 16S rRNA gene sequence similarity). We propose a novel genus and species, Chryseolinea serpens gen. nov., sp. nov.. Strain RYGT ( = DSM 24574T = ATCC BAA-2075T) is the type strain.

A strictly aerobic, Gram-stain-negative, yellow-pigmented, non-spore-forming, motile (by gliding), rod-shaped bacterium, designated strain 15F3 T , was isolated from leaf-and-branch compost. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 15F3T was most closely related to Flavobacterium reichenbachii WB 3.2-61 T and formed a distinct phyletic lineage within the genus Flavobacterium, the type genus of the family Flavobacteriaceae. Growth was observed at 10-34 6C (optimum, 30 6C) and pH 6.0-8.0 (optimum, pH 7.0). No growth occurred in the presence of ¢2 % (w/v) NaCl. Strain 15F3 T reduced nitrate to nitrogen and showed catalase activity but no oxidase activity. The predominant cellular fatty acids were iso-C 15 : 0 and summed feature 3 (comprising C 16 : 1 v7c and/or iso-C 15 : 0 2-OH). The major isoprenoid quinone was menaquinone-6. The G+C content of the genomic DNA was 31.1 mol%. On the basis of data from this polyphasic study, strain 15F3 T may be classified as a representative of a novel species within the genus Flavobacterium, for which the name Flavobacterium banpakuense sp. nov. is proposed; the type strain is 15F3 T (5KACC 14225The genus Flavobacterium is the type genus of the family Flavobacteriaceae in the phylum 'Bacteroidetes'. Species of the genus Flavobacterium are defined as Gram-stainnegative, aerobic, yellow-pigmented, rod-shaped bacteria that are non-motile or motile by gliding. DNA G+C contents range from 30 to 52 mol% and menaquinone-6 (MK-6) is the major respiratory quinone (Bernardet & Bowman, 2006;Liu et al., 2010). Strains representing the genus Flavobacterium have been found in a variety of environments such as soil, freshwater, seawater, sediments and diseased fish (Cousin et al., 2007; Yoon et al., 2009 et al., 2008). They show very diverse physiological characteristics being either psychrophilic or mesophilic and either halophilic or sensitive to salts. The variety of enzymes they produce, some of them having potential applications in industry and bioremediation (Humphry et al., 2001;Tamaki et al., 2003;Riffel & Brandelli, 2002;Singh, 2009), suggests that they have important roles in the environment.Most strains of the genus Flavobacterium are able to degrade polysaccharides such as carboxymethyl-cellulose (McCammon & Bowman, 2000;Bernardet et al., 1996;Park et al., 2007), xylan (Lee et al., 2006), chitin (Bernardet et al., 1996) and b-1,3-glucan (Rasmussen et al., 2008). For instance, Flavobacterium johnsoniae has been shown to degrade chitin and CM-cellulose (Reichenbach, 1989;Bernardet et al., 1996;McBride et al., 2003). Here, the isolation and identification of a novel polysaccharidedegrading (e.g. starch and CM-cellulose) strain that represents a novel species in the genus Flavobacterium are reported. Strain 15F3T was isolated from aerobic leaf-and-branch compost at EXPO Park in Osaka, Japan. The compost was made from leaves and branches cut from trees. These leaves and branches were piled up in an open space to 2 m high. Urea was added to the stack as a...

HighlightsTen novel cellulases, LC-CelA–J, were isolated from leaf–branch compost by a metagenomic approach.LC-CelA was characterized.The structure, activity, and stability of LC-CelA were similar to those of Cel12A from Rhodothermus marinus.Glu34-mediated hydrogen bonds and two disulfide bonds contribute to the stabilization of LC-CelA.

A strictly aerobic, Gram-negative, yellow-pigmented, non-spore-forming rod, designated 15C3 T , was isolated from aerobic leaf-and-branch compost at EXPO Park in Osaka, Japan. Growth was observed at 9-33 6C (optimum 25 6C) and pH 5.6-7.9 (optimum pH 6.1-7.0). No growth occurred with .2 % (w/v) NaCl. Strain 15C3 T reduced nitrate to nitrogen and showed catalase activity but not oxidase activity. The predominant fatty acids were iso-C 15 : 0 , iso-C 17 : 0 3-OH and summed feature 3 (comprising C 16 : 1 v7c and/or iso-C 15 : 0 2-OH). The isolate contained phosphatidylethanolamine as the major polar lipid and menaquinone-6 as the major respiratory quinone. The G+C content of the genomic DNA of strain 15C3 T was 33.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 15C3 T belonged to the genus Flavobacterium and was most closely related to Flavobacterium hercynium WB 4.2-33 T (96.9 % sequence similarity). On the basis of phenotypic and phylogenetic distinctiveness, strain 15C3 T is considered to represent a novel species in the genus Flavobacterium, for which the name Flavobacterium compostarboris sp. nov. is proposed. The type strain is 15C3 T (5KACC 14224 T 5JCM 16527 T ). Emended descriptions of F. hercynium, Flavobacterium resistens and Flavobacterium johnsoniae are also given.The genus Flavobacterium, which belongs to the phylum 'Bacteroidetes', is the type genus of the family Flavobacteriaceae. Members of the genus are Gram-negative, aerobic, yellow-pigmented rods that are predominantly motile by gliding, contain menaquinone-6 (MK-6) as the major respiratory quinone and have a DNA G+C content in the range 30-52 mol% (Bernardet et al., 1996;Bernardet & Bowman, 2006Liu et al., 2010).Along with the genera Chitinophaga, Pedobacter and Chryseobacterium, the genus Flavobacterium has shown one of the highest increases in the number of species for the family Flavobacteriaceae. Recently, many novel species of the genus Flavobacterium have been isolated from a variety of habitats, such as soil (Lim et al., 2010;Yang et al., 2011), freshwater (Sheu et al., 2011), seawater (Yoon et al., 2011), sediments (Fu et al., 2011Lee et al., 2010), glacier-frozen soil (Xu et al., 2011), wastewater treatment systems (Zhang et al., 2010;Liu et al., 2010), marine algae (Miyashita et al., 2010), rhizospheres (Xiao et al., 2011;Madhaiyan et al., 2010), leaf-andbranch compost (Kim et al., 2011 and reed roots . Some strains produce enzymes that are able to degrade a variety of biopolymers and/or are active at low temperatures . These enzymes have a potential use in biotechnological applications.The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain 15C3 T is GQ281769.A supplementary figure is available with the online version of this paper. In a study of cellulose-degrading bacteria, a Gram-negative CM-cellulose-degrading rod was isolated from aerobic leafand-branch compost at EXPO Park in Osaka, Japan. Strain 15C3 T was isolated from 4-month-old, spontaneously fermented compost (pH ...

BackgroundSymbiobacterium toebii is a commensal symbiotic thermophile that absolutely requires its partner bacterium Geobacillus toebii for growth. Despite development of an independent cultivation method using cell-free extracts, the growth of Symbiobacterium remains unknown due to our poor understanding of the symbiotic relationship with its partner bacterium. Here, we investigated the interrelationship between these two bacteria for growth of S. toebii using different cell-free extracts of G. toebii.ResultsSymbiobacterium toebii growth-supporting factors were constitutively produced through almost all growth phases and under different oxygen tensions in G. toebii, indicating that the factor may be essential components for growth of G. toebii as well as S. toebii. The growing conditions of G. toebii under different oxygen tension dramatically affected to the initial growth of S. toebii and the retarded lag phase was completely shortened by reducing agent, L-cysteine indicating an evidence of commensal interaction of microaerobic and anaerobic bacterium S. toebii with a facultative aerobic bacterium G. toebii. In addition, the growth curve of S. toebii showed a dependency on the protein concentration of cell-free extracts of G. toebii, demonstrating that the G. toebii-derived factors have nutrient-like characters but not quorum-sensing characters.ConclusionsNot only the consistent existence of the factor in G. toebii during all growth stages and under different oxygen tensions but also the concentration dependency of the factor for proliferation and optimal growth of S. toebii, suggests that an important biosynthetic machinery lacks in S. toebii during evolution. The commensal symbiotic bacterium, S. toebii uptakes certain ubiquitous and essential compound for its growth from environment or neighboring bacteria that shares the equivalent compounds. Moreover, G. toebii grown under aerobic condition shortened the lag phase of S. toebii under anaerobic and microaerobic conditions, suggests a possible commensal interaction that G. toebii scavengers ROS/RNS species and helps the initial growth of S. toebii.