Cytochrome cd1-containing nitrite reductase encoding gene nirS as a new functional biomarker for detection of anaerobic ammonium oxidizing (Anammox) bacteria

A newly designed primer set (AnnirS), together with a previously published primer set (ScnirS), was used to detect anammox bacterial nirS genes from sediments collected from three marine environments. Phylogenetic analysis demonstrated that all retrieved sequences were clearly different from typical denitrifiers' nirS, but do group together with the known anammox bacterial nirS. Sequences targeted by ScnirS are closely related to Scalindua nirS genes recovered from the Peruvian oxygen minimum zone (OMZ), whereas sequences targeted by AnnirS are more closely affiliated with the nirS of Candidatus 'Kuenenia stuttgartiensis' and even form a new phylogenetic nirS clade, which might be related to other genera of the anammox bacteria. Analysis demonstrated that retrieved sequences had higher sequence identities (>60%) with known anammox bacterial nirS genes than with denitrifiers' nirS, on both nucleotide and amino acid levels. Compared to the 16S rRNA and hydrazine oxidoreductase (hzo) genes, the anammox bacterial nirS not only showed consistent phylogenetic relationships but also demonstrated more reliable quantification of anammox bacteria because of the single copy of the nirS gene in the anammox bacterial genome and the specificity of PCR primers for different genera of anammox bacteria, thus providing a suitable functional biomarker for investigation of anammox bacteria.     

Molecular cloning and characterization of a glucan synthase gene from the human pathogenic fungus Paracoccidioides brasiliensis

1,3-beta-D-glucan is a fungal cell wall polymer synthesized by the multi-subunit enzyme 1,3-beta-D-glucan synthase. A subunit of this integral membrane protein was first described as the product of the FKS1 gene from Saccharomyces cerevisiae using echinocandin mutants. Other FKS1 genes were also reported for Candida albicans, Aspergillus nidulans and Cryptococcus neoformans. Here, we report the nucleotide sequence of the first homologous FKS gene cloned from the pathogenic fungus Paracoccidioides brasiliensis. An open reading frame of 5942 bp was identified in the complete sequence, interrupted by two putative introns, the first close to the 5' end and the second close to the 3' end of the gene. A promoter region is also described containing consensus sequences such as canonical TATA and CAAT boxes and, possibly, multiple sites for glucose regulation by creA protein. The deduced sequence of 1926 amino acid show more than 85% similarity to FksAp from A. nidulans, and 71% to Fks1p and Fks2p from S. cerevisiae. Computational analysis of P. brasiliensis Fks1p suggests a similar structure to transmembrane proteins, such as FksAp, with the presence of two domains composed by hydrophobic helices that limit the putative highly hydrophilic catalytic domain within the cytoplasm.     

Enhancement and inhibition of denitrification by fluid-flow and dissolved oxygen flux to stream sediments

Microscale measurements of nitrate (NO3-) and dissolved oxygen (DO) concentrations in sediments were made in a laboratory channel under turbulent fluid-flow conditions to examine the effects of DO flux on denitrification rates. DO concentrations and flux within sediments increased with increasing velocity in the surface water. Under low fluid-flow conditions (shear stress velocity, u* < 0.23 cm s(-1)), increasing velocity increased NO3- loss from the bulk flow. For high fluid-flow conditions (u* > 0.39 cm s(-1)), increasing velocity inhibited NO3-loss. Sediment cores were collected and sliced to measure the depth distribution of denitrifying biomass in sediments. Quantities of nirK and nirS genes were higher within the surface layer and decreased with depth in the sediments. Microscale concentration profiles of DO and NO3- revealed that denitrification occurs within a thin region just below the oxic-anoxic interface in sediments. The interplay of mass transfer and DO flux generated threshold conditions for NO3- loss by denitrification. These results suggest that for a given sediment and environmental conditions (chemical, physical, microbiological), there exists an optimal range in velocities for enhancing denitrification in aquatic systems.     

Characterization of denitrifying polyphosphate-accumulating organisms in activated sludge based on nitrite reductase gene

Nitrite reductase gene (nirS) fragments in the activated sludge obtained from a sequencing batch reactor (SBR) under anaerobic-aerobic condition were cloned and classified by restriction fragment length polymorphism (RFLP) analysis, and representative fragments were sequenced. One of the nirS clones was approximately 70% of all nirS clones in anaerobic/aerobic (existing oxygen and nitrate) cycle operation in which a large amount of anoxic phosphate uptake was observed. Although the activated sludge samples analyzed might contain bacteria that did not accumulate polyphosphate, it was likely that this nirS fragment sequence was that from denitrifying polyphosphate-accumulating organisms (DNPAOs) which can utilize both oxygen and nitrate as electron acceptors. The sequence was similar to the nirS sequences of Thauera mechernichensis (83% similarity) and Azoarcus tolulyticus (83% similarity) both of which belong to the Rhodocyclus group.     

Identification of species in Aspergillus section Flavi based on sequencing of the mitochondrial cytochrome b gene.

The partial sequences of the mitochondrial (mt) cytochrome b gene (402 bp) were determined for species of Aspergillus section Flavi. On the basis of identities of DNA sequences, 77 strains were divided into seven DNA types, from D-1 to D-7. The type strains of A. sojae, A. parasiticus, A. flavus and A. oryzae together, A. tamarii, and A. nomius were placed in DNA types D-1. D-2, D-4, D-5 and D-7, respectively. These species could be differentiated from each other. Furthermore, two other DNA types, D-3 and D-6 were found. DNA type D-3 was closely related to A. parasiticus (D-2) and included one strain that deposited as A. flatus var. flavus and produced aflatoxins B and G. DNA types D-6 included one strain named A. flavus and closely related to A. tamarii. The observations of conidial wall texture by SEM (Scanning Electron Microscopy) supported the relationships derived from the cytochrome b gene. The production of aflatoxins was also examined. Using the DNA sequence of cytochrome b gene, several strains were reidentified. The derived amino acids sequences were all the same in the studied strains. The mt cytochrome b gene is useful and reliable in distinguishing and identifying the species in Aspergillus section Flavi.     

Cloning and nucleotide sequence of carbaryl hydrolase gene (cahA) from Arthrobacter sp. RC100

A carbaryl hydrolase gene (cahA) encoded on the plasmid pRC1 in Arthrobacter sp. RC100 was cloned and sequenced. The entire region of the deduced amino acid sequence was found to be homologous to that of an amidase family. Parts of the consensus sequences of the amidase gene have been identified in CahA from strain RC100. CahA was overexpressed in Escherichia coli JM109, and the enzyme was purified to homogeneity by protamine sulfate treatment, ammonium sulfate precipitation, and hydrophobic and anion-exchange chromatographies. The purified enzyme showed hydrolase activity toward 1-naphthylacetamide and isobutyramide but showed no activity toward 1-naphthylacetate. This is the first report of an amidase that is able to hydrolyze N-methylcarbamate pesticides.     

Characterization of the cytochrome c gene from the starch-fermenting yeast Schwanniomyces occidentalis and its expression in Baker's yeast

A cytochrome c protein gene, CYC10, of the dextran- and starch-fermenting yeast, Schwanniomyces occidentalis was cloned and characterized. The DNA sequence was determined, and the predicted amino acid sequence of the protein-coding region shares close homologies to the cytochrome c genes. A S. occidentalis strain with a disruption of the gene revealed that CYC10 was the only functional cytochrome c protein-encoding gene in S. occidentalis, unlike the two cytochrome c protein genes (CYC1 and CYC7) in Saccharomyces cerevisiae. The CYC10 gene was oxygen-induced but not subject to catabolite repression. The expression of the CYC10 gene was studied in the heterologous yeast S. cerevisiae. The oxygen induction of the gene was found to be identical to that of the CYC1 gene, indicating that these two genes share similar or closely related cis- and trans-acting oxygen regulatory elements. However, the CYC10 gene was glucose repressed in S. cerevisiae strains; a phenomenon which was not observed in the native S. occidentalis cells. Search in the 5' untranslated region of the CYC10 gene revealed some homologies at -425 to -405 to UAS1 of the S. cerevisiae CYC1 gene. A deletion of a segment of upstream region including this sequence abolished expression in S. cerevisiae. Finally the phylogenetic relationships of different yeasts and fungi were determined based upon the amino acid sequences of the cytochrome c proteins. These relationships do not completely agree with classical divisions.     

Cloning and sequencing of an exoglucanase gene from Streptomyces sp. M 23, and its expression in Streptomyces lividans TK-24

A gene encoding exoglucanase (CBHII) of Streptomyces sp. M 23 was cloned and sequenced. The cbhII gene consisted of 1359 bp capable of encoding a polypeptide of 453 amino acids with a calculated molecular mass of 45,175 Da. The deduced amino acid sequence showed homology with those of cellulases belonging to family 6 of the glycosyl hydrolases. The cbhII gene was subcloned into the plasmid pSEV1 and expressed in Streptomyces lividans TK-24. The transformed cells were able to secrete the enzyme efficiently in an active form. The CBHII expressed by S. lividans TK-24 was purified to homogeneity by SDS-polyacrylamide gel and characterized. The recombinant CBHII was stable up to 50 degrees C and more than 30% of the original activity remained after heating at 100 degrees C for 10 min. The amino-terminal amino acid sequence of the recombinant CBHII was identified as GPAAPTARVD. These results agreed well with the properties of the authentic CBHII.     

Analysis of DNA sequences homologous with the ARS core consensus in Saccharomyces cerevisiae

We have previously identified an autonomously replicating segment (ARS) near the 3' end of the histone H4 gene at the copy-I H3-H4 locus. We have now searched for additional autonomously replicating segments and sequences homologous with the ARS core consensus sequence near the copy-II histone H4 gene and both of the histone H3 genes. No new ARS elements were identified by functional cloning assays. However, several matches to the ARS core consensus element were found within the DNA sequences of the copy-I and copy-II genes. An exact match to the ARS core consensus was identified in the region downstream from the copy-I histone H3 gene and a set of sequences with weak homology was also located within the copy-II region. However, restriction fragments including these sequences did not demonstrate ARS activity on a plasmid in transformed cells.     

Characterization and crystal structure of the thermophilic ROK hexokinase from Thermus thermophilus

We characterized and determined the crystal structure of a putative glucokinase/hexokinase from Thermus thermophilus that belongs to the ROK (bacterial repressors, uncharacterized open reading frames, and sugar kinases) family. The protein possessed significant enzymatic activity against glucose and mannose, with V(max) values of 260 and 68 μmol·min(-1)·mg(-1) protein, respectively. Therefore, we concluded that the enzyme is a hexokinase. However, the hexokinase showed little catalytic capacity for galactose and fructose. Circular dichroism measurements indicated that the enzyme was structurally stable at 90°C. The crystal structure of the enzyme was determined at a resolution of 2.02 ?, with R(cryst) and R(free) values of 18.1% and 22.6%, respectively. The polypeptide structure was divided into large and small domains. The ROK consensus sequences 1 and 2 were included in the large domain. The cysteine-rich consensus sequence 2 folded into a zinc finger, and the bound zinc was confirmed by both electron density and X-ray absorption fine structure (XAFS) spectrum. The overall structure was a homotetramer that consisted of a dimer of dimers. The accessible surface area buried by the association of the dimers into the tetrameric structures was significantly higher in the T. thermophilus enzyme than in a homologous tetrameric ROK sugar kinase.     

Analysis of the MUC1 gene and its polymorphism in Capra hircus

The objective of our study was to demonstrate the existence of a repetitive region in the goat MUC1 gene and to develop a polymerase chain reaction (PCR) protocol to analyze its polymorphism in different breeds. Using 2 primers derived from the bovine MUC1 sequence, a PCR fragment was obtained and cloned. The sequence analysis shows that the repetitive region of goat MUC1 is an array of 60 bp repeats in accordance with the data reported for other species. The polypeptide sequences encoded by the consensus repeats of goat and bovine were exactly alike. A PCR protocol to improve the detection of goat MUC1 polymorphism was developed, and a total of 178 animals from 6 Italian breeds were analyzed. Fifteen different alleles, ranging in size from 1500 to 3000 bp, were found. The high number of alleles observed shows that the goat MUC1 is a hypervariable gene. These results are the basis for further investigations on the possible role of MUC1 polymorphism in the genetic control of disease susceptibility and production traits in the goat species.     

Molecular cloning and characterization of a novel bacteriophage-associated sialidase

Bacteriophage 63D, previously isolated from sewage, is associated with alpha-2,8-linked polysialic acid degrading activity. We cloned a DNA fragment containing the sialidase gene from a 63D phage genomic library and the enzyme was functionally expressed in Escherichia coli. Determination of the nucleotide sequence of the fragment revealed that it contained one open reading frame (ORF) coding for a 108-kDa polypeptide consisting of 984 amino acid residues. The fragment had promoter sequences similar to the E. coli consensus promoters for sigma70. The deduced amino acid sequence of the central region of the ORF showed homology to those of phages K1F (51.6% identity) and PK1E (51.7% identity) endosialidases. Two Asp-box motifs that are widely found in sialidases were conserved. Purification of the soluble enzyme from lysed culture broth of infected E. coli yielded a 90-kDa protein upon SDS polyacrylamide gel electrophoresis, suggesting that the primary translational product is processed to the mature 90-kDa protein. The molecular mass of the enzyme was determined as 360 kDa by gel filtration, indicating that the native enzyme was probably a tetramer of identical 90-kDa subunits.     

大豆rbcL基因克隆、序列分析及原核表达

利用叶绿体基因组保守性的特征,根据菜豆、豌豆、烟草的rbcL基因序列设计引物,从大豆叶绿体DNA中克隆rbcL基因,全长序列为1488bp,包括1449bp的开放阅读框,编码482个氨基酸。相似性比较显示,此序列与其它10个物种rbcL基因核苷酸的同源性为85．37％～95．31％,氨基酸的同源性为90．87％-96．47％。将该基因与表达载体pET-30a（＋）连接,转化大肠杆菌Rosseta感受态细胞,PCR和酶切鉴定筛选阳性克隆,阳性菌液IPTG诱导后经10％SDS—PAGE分析,结果显示,诱导表达出分子量约为60kD的特异融合蛋白。     

Denitrifying polyphosphate accumulating organisms population and nitrite reductase gene diversity shift in a DEPHANOX-type activated sludge system fed with municipal wastewater

Enhanced biological phosphorus removal (EBPR) is a widely applied method for nutrients removal, although little is known about the key genes regulating the complex biochemical transformations occurring in activated sludge during phosphorus removal. In the present study, the nitrite reductase gene (nirS) diversity and the denitrifying polyphosphate accumulating organisms (DPAOs) population, grown in a bench scale, two-sludge, continuous flow plant, operating for biological anoxic phosphorus removal (DEPHANOX-type), fed with municipal wastewater, were examined by means of physicochemical analyses and the application of molecular techniques. The DEPHANOX configuration highly influenced biomass phosphorus as well as polyhydroxyalkanoates content and facilitated the enrichment of the DPAOs population. The application of double probe fluorescent in situ hybridization (double probe FISH) technique revealed that DPAOs comprised 20% of the total bacterial population. Based on clone libraries construction and nirS gene sequencing analysis, a pronounced shift in denitrifying bacteria diversity was identified during activated sludge acclimatization. Moreover, nirS gene sequences distinct from those detected in any known bacterial strain or environmental clone were identified. This is the first report studying the microbial properties of activated sludge in a DEPHANOX-type system using molecular techniques.     

Purification, characterization, and steady-state kinetics of a meta-cleavage compound hydrolase from Pseudomonas fluorescens IPO1

2-Hydroxy-6-oxo-7-methylocta-2,4-dienoate (6-isopropyl-HODA) hydrolase (CumD), an enzyme of the cumene biodegradation pathway encoded by the cumD gene of Pseudomonas fluorescens IP01, was purified to homogeneity from an overexpressing Escherichia coli strain. SDS-polyacrylamide gel electrophoresis and gel filtration demonstrated that it is a dimeric enzyme with a subunit molecular mass of 32 kDa. The pH optima for activity and stability were 8.0 and 7.0-9.0, respectively. The enzyme exhibited a biphasic Arrhenius plot of catalysis with two characteristic energies of activation with a break point at 20 degrees C. The enzyme has a K(m) of 7.3 microM and a k(cat) of 21 s(-1) for 6-isopropyl-HODA (150 mM phosphate, pH 7.5, 25 degrees C), and its substrate specificity covers larger C6 substituents compared with another monoalkylbenzene hydrolase, TodR Unlike TodF, CumD could slightly hydrolyze 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (6-phenyl-HODA). A mutant enzyme as to a putative active site residue, S103A, had 10(5)-fold lower activity than that of the wild-type enzyme.