We have recently reported the purification and characterization of a novel esterase from the Bacillus subtilis strain. In the present study we report the genomic DNA cloning and predictive structural modeling of this novel esterase. Tributyrin- and Rhodamine B-based functional screen of a Bacillus subtilis genomic library led to the identification of a potential lipolytic gene. DNA sequence analysis of the cloned gene showed that it encodes a protein of 489 amino acid residues. Sequence homology search and multiple sequence alignment showed that the protein was highly homologous to known esterases. Secondary structure-driven multiple sequence alignment with the homologous esterase of known three-dimensional structures was performed and a 3D structure model of this enzyme was constructed. Based on the topological organization of the secondary structures, this protein belongs to the α/β hydrolase superfamily. Moreover, the presence of serine in the context of amino acid sequence G/A-X-S-X-G (with X an arbitary amino acid residue) in the protein indicates that it belong to the class of serine hydrolases of this superfamily.
A novel extremely thermophilic bacterium (Ni80T) was isolated from food sludge compost in Japan. Cells were Gram-positive, spore-forming, strictly-aerobic and rod-shaped with peritrichous flagella. The novel isolate grew at pH 6.5-10.5 but not at pH 11.0 (optimum pH 8.0) and 52-79°C but not 80°C (optimum 70°C). The strain could not grow in more than 1% NaCl. The G+C content of the genomic DNA is 72 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain Ni80T belonged to the genus Thermaerobacter, and that the strain was most closely related to Thermaerobacter subterraneus ATCC BAA-137T (98.2%), Thermaerobacter nagasakiensis JCM 11223T (98.2%), Thermaerobacter marianensis JCM 10246T (98.0%) and Thermaerobacter litoralis JCM 13210T (97.7%). DNA-DNA hybridization showed relatedness values of less than 10% with these species. On the basis of phenotypic and molecular data, strain Ni80T represent a novel species of the genus Thermaerobacter, for which the name Thermaerobacter composti sp. nov. is proposed. The type strain is Ni80T (=JCM 15650T =ATCC BAA-1731T).
Soil bacterial community compositions in burnt and unburnt areas in a tropical rainforest in East Kalimantan, Indonesia, were investigated 8 and 9 years after a fire by denaturing gradient gel electrophoresis analysis targeting the 16S rRNA gene. Three study sites were set in the forest area devoid of fire damage (control), and in the lightly damaged and heavily damaged forest areas. Succession of aboveground vegetation in the two damaged areas had clearly proceeded after the fire, but the vegetation types still differed from the unburnt area at the time of this study. Community composition of total soil bacteria was similar among the three areas, and so was that of actinobacteria. However, the composition of ammonia oxidizing bacteria clearly differed depending on the presence or absence of past fire damage. These results indicate that even nearly a decade after the forest fire, impacts of the fire remained on the community composition of ammonia oxidizing bacteria, but not apparently on those of dominant bacteria and actinobacteria.
An actinobacterial strain, 2SbT, isolated from lichen was characterized taxonomically using a polyphasic approach. Strain 2SbT was Gram-positive, strictly aerobic, rod-shaped and non-motile. Phylogenetic analysis based on 16S rRNA gene sequence revealed that strain 2SbT was located in the genus Leifsonia. Level of 16S rRNA gene sequence similarity between the isolated strains and the type strains of Leifsonia species were 95.0-99.2%. The value of DNA-DNA relatedness between strain 2SbT and Leifsonia poae, a phylogenetically related and phenotypically similar species, was 39.9/25.2%. The DNA G+C content of the strain 2SbT was 61.7 mol%. Cell wall peptidoglycan type (2,4-diaminobutyric acid), major cellular fatty acids (anteiso-C15:0, anteiso-C17:0 and iso-C16:0) and quinone type (MK-11 and MK-10) of the isolate support their affiliation to the genus Leifsonia. On the basis of phylogenetic position, physiological and chemotaxonomic properties, strain 2SbT represent a novel species of the genus Leifsonia, for which the name Leifsonia lichenia sp. nov. is proposed. The type strain is 2SbT (=IAM 15426T =JCM 23226T =KCTC 13122T ).
Citrobacter braakii produced an intracellular acid glucose phosphatase (AgpC) which was purified 986 fold to homogeneity with the specific activity of 286 units/mg. AgpC hydrolyzed a wide variety of phosphorylated compounds with high activity for glucose-1-phosphate and glucose-6-phosphate. The optimum pH and temperature for the enzyme activity was pH 5.0 and 45°C, respectively. The Km value for glucose-1-phosphate was 5.12 mM with a Vmax 27.8 U mg-1. Its molecular weight was 46 kDa by SDS-PAGE gel and the sequence of N-terminal amino acid residues identified was Gln-Thr-Ala-Pro-Glu-Gly-Tyr-Gln-Leu-Gln. The glucose-1-phosphatase gene (agpC) was cloned from the C. braakii genomic library. This gene comprised 1,242 nucleotides and encoded a polypeptide of 413 amino acids. The result of its BLAST search showed a significant similarity with glucose-1-phosphatase from enterobacteria such as E. coli, Enterobacter, Shigella, and Salmonella.
Recent studies have shown that the ferric uptake regulator (Fur) of Edwardsiella tarda (FurEt) shares high sequence identity with the Escherichia coli Fur (FurEc) at the N-terminal DNA-binding region. In the present study, the functional importance of the C-terminal region of FurEt was investigated. It was found that FurEt bearing deletion of the C-terminal 12 residues still possesses most of the repressor activity, whereas FurEt bearing deletions of the C-terminal 16 and more than 16 residues are severely affected in activity. Domain swapping analyses indicated that the chimeric Fur proteins (Et75Ec73 and Et75Vh74) consisting of the N-terminal 1-75 region of FurEt fused to the C-terminal 76-148 region of FurEc and the C-terminal 76-149 region of the Vibrio harveyi Fur (FurVh), respectively, are fully active. C92 of FurEc and C137 of FurVh, which are functionally essential in FurEc and FurVh, respectively, are also essential in Et75Ec73 and Et75Vh74, respectively. Further study identified an artificial Fur protein, EtMF54, which is composed of the N-terminal 49 residues of FurEt and five artificial residues. Compared to FurEt, EtMF54 possesses partial Fur activity that is iron-dependent. These results (i) indicate that there exist certain functional/structural compatibilities among FurEt, FurEc, and FurVh at the C-terminal region; (ii) provide insights to the potential location of the regulatory ion-binding site of FurEt.
Bacterial populations in epilithic biofilms collected from two distinct oligotrophic rivers of Japan were studied using denaturing gradient gel electrophoresis (DGGE). PCR-DGGE of the 16S rRNA gene and subsequent sequencing analysis suggested that in freshwater biofilms, members of the Cytophaga-Flavobacterium-Bacteroides (CFB) group were the most dominant, followed by those of α, β, γ, and δ-Proteobacteria; Leptospiraceae; and unidentified bacteria. Members of the CFB group, α-Proteobacteria, and cyanobacteria/plastid DNA were also detected from the biofilms collected from the estuary site, but the species in these samples differed from those detected in biofilms in the freshwater areas of the rivers. A comparison between the determined sequences revealed that similar bacterial species existed in biofilms at different sites of a river, and identical species existed in biofilms of distinct rivers. The results suggested that bacterial species in biofilms found in the estuary were different from those found in the freshwater areas of the rivers; however, the common bacterial species were distributed in biofilms collected from not only different sites along the same river but also sites in distinct oligotrophic rivers.
Biological treatment based on activated sludge is efficient in municipal wastewater treatment. The removal of nitrogen compounds is crucial for sewage purification. Due to the fact that ammonia is toxic for a water environment and causes eutrophication, ammonia oxidation is of the utmost importance in wastewater treatment. Observing the changeability of ammonia oxidizing bacteria (AOB) and identifying their most abundant species can be helpful in the optimization of wastewater treatment. In this study we used denaturing gradient gel electrophoresis (DGGE), combined with cloning and sequencing of 16S rRNA and AmoA gene fragments in order to estimate AOB biodiversity and temporal community changes. Activated sludge samples were collected from the municipal WWTP in Gliwice (Poland) at 2-week intervals. Ammonia concentration in the influent during the experiment was 30.2-57.6 mg N-NH4+/L. The research revealed a high diversity of uncultured bacteria. It is suspected that these bacteria could be involved in the nitrification, which points to the fact that these bacteria might be efficient in the process. However such a situation is not confirmed and it requires further research. The appearance of Ferribacterium-like bacteria together with Nitrosomonas sp. as the most abundant bacteria was found.
Computational approaches provide valuable information to start experimental surveys identifying glycosylphosphatidylinositol (GPI)-anchored proteins in protein sequence databases. We developed a new sequence-based identification system that uses an optimized classifier based on a support vector machine (SVM) algorithm to recognize appropriate COOH-terminal sequences and uses a classifier implementing a simple majority voting strategy to recognize appropriate NH2-terminal sequences. The SVM classifier showed high accuracy (96%) in 5-fold cross-validation testing, and the majority voting classifier showed high recall (98.88%) when applied to a test dataset of eukaryote proteins. When applied to S. cerevisiae protein sequences, the new identification system showed good ability to classify “unseen” data. Applying our system to protein sequences of three aspergilli, we identified 115 GPI-anchored proteins in Aspergillus fumigatus, 129 in Aspergillus nidulans, and 136 in Aspergillus oryzae. Sequence-based conserved domain search found nearly half of these proteins to have conserved domains that covered a wide range of functions.
Human α-defensin 5 (HD5), a small cationic peptide, is expressed in Paneth cell granules of small intestinal crypts. HD5 exhibits high antimicrobial activity against a broad spectrum of pathogenic agents, including bacteria, fungi, and viruses. In this study, the constitutive expression of HD5 antimicrobial peptide was achieved using the methylotrophic yeast, Pichia pastoris (P. pastoris). HD5 cDNA was amplified by polymerase chain reaction (PCR) using human lung cell cDNA as template. The 96-bp DNA fragment encoding mature HD5 peptide (amino acid 63-94) was subcloned into the yeast expression vector and transfected into P. pastoris X-33 expression host by electroporation. The recombinant HD5 (rHD5) was detected in the supernatant of transfected yeast by western blot analysis. The recombinant HD5 crude extract from transfected P. pastoris showed antimicrobial activities against Salmonella typhimurium, Stapheloccus aureus and pathogenic E. coli. However, rHD5 did not inhibit the growth of lactic acid bacteria such as Lactobacillus bulgaricus, Bifidobacterium bifidum, or B. longum. These results indicated that the rHD5 expressed in P. pastoris selectively inhibited the growth of specific bacteria.