Structural and Functional Characterization of a New Bacterial Dipeptidyl Peptidase III Involved in Fruiting Body Formation in Myxobacteria.

Dipeptidyl peptidase III (DPP III) is a zinc-dependent enzyme that specifically hydrolyzes dipeptides from the N-terminal of different-length peptides, and it is involved in a number of physiological processes. Here, DPP III with an atypical pentapeptide zinc binding motif (HELMH) was identified from Corallococcus sp. EGB. It was shown that the activity of recombined CoDPP III was optimal at 50 °C and pH 7.0 with high thermostability up to 60 °C. Unique to CoDPP III, the crystal structure of the ligand-free enzyme was determined as a dimeric and closed form. The relatively small inter-domain cleft creates a narrower entrance to the substrate binding site and the unfavorable binding of the bulky naphthalene ring. The ectopic expression of CoDPP III in M. xanthus DK1622 resulted in a 12 h head start in fruiting body development compared with the wild type. Additionally, the A-signal prepared from the starving DK1622-CoDPP III rescued the developmental defect of the asgA mutant, and the fruiting bodies were more numerous and closely packed. Our data suggested that CoDPP III played a role in the fruiting body development of myxobacteria through the accumulation of peptides and amino acids to act as the A-signal.

Characterization of Stackebrandtia nassauensis GH 20 Beta-Hexosaminidase, a Versatile Biocatalyst for Chitobiose Degradation.

An unstudied ß-N-acetylhexosaminidase (SnHex) from the soil bacterium Stackebrandtia nassauensis was successfully cloned and subsequently expressed as a soluble protein in Escherichia coli. Activity tests and the biochemical characterization of the purified protein revealed an optimum pH of 6.0 and a robust thermal stability at 50 °C within 24 h. The addition of urea (1 M) or sodium dodecyl sulfate (1% w/v) reduced the activity of the enzyme by 44% and 58%, respectively, whereas the addition of divalent metal ions had no effect on the enzymatic activity. PUGNAc (O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate) strongly inhibited the enzyme in sub-micromolar concentrations. The ß-N-acetylhexosaminidase was able to hydrolyze ß1,2-linked, ß1,3-linked, ß1,4-linked, and ß1,6-linked GlcNAc residues from the non-reducing end of various tested glycan standards, including bisecting GlcNAc from one of the tested hybrid-type N-glycan substrates. A mutational study revealed that the amino acids D306 and E307 bear the catalytically relevant side acid/base side chains. When coupled with a chitinase, the ß-N-acetylhexosaminidase was able to generate GlcNAc directly from colloidal chitin, which showed the potential of this enzyme for biotechnological applications.

Taonella mepensis gen. nov., sp. nov., a member of the family Rhodospirillaceae isolated from activated sludge.

A novel Gram-negative, non-spore-forming, rod-shaped strain, H1(T), was isolated from activated sludge by micromanipulation. No close relatives among cultured bacterial isolates were found; phylogenetic analysis based on 16S rRNA gene sequences revealed that strain H1(T) forms a deep single branch in the family Rhodospirillaceae. Cells of strain H1(T) were slightly curved to straight rods (1.2-1.4 × 1.5-1.7 µm) and motile by a single polar flagellum. Strain H1(T) was able to grow in the presence of 0-4 % NaCl and grew optimally at 37 °C and pH 6.0-7.0. Chemotaxonomic analysis revealed that strain H1(T) possessed Q-10 as the predominant ubiquinone and C18 : 1ω7c, C16 : 0 and C19 : 0 cyclo ω8c as the major fatty acids. The DNA G+C content of strain H1(T) was 65.1 mol%. Comparative analysis of 16S rRNA gene sequences, and phenotypic and chemotaxonomic data, indicate that strain H1(T) should represent a novel genus and species of the family Rhodospirillaceae. The name Taonella mepensis gen. nov., sp. nov. is proposed. The type strain of Taonella mepensis is H1(T) ( = CICC 10529(T) = CCTCC AB 2012861(T) = KACC 16940(T)).

Cloning and functional characterization of a novel endo-ß-1,4-glucanase gene from a soil-derived metagenomic library.

A metagenomic library consisting of 3,024 bacterial artificial chromosome clones was prepared in Escherichia coli DH10B with high-molecular-weight DNA extracted from red soil in South China. A novel cellulase gene with an open reading frame of 1,332 bp, cel5G, encoding an endo-ß-1,4-glucanase was cloned using an activity-based screen. The deduced enzyme, Cel5G, belongs to the glycosyl hydrolase family 5 and shares <39% identity with endoglucanases in the GenBank database. cel5G was expressed in E. coli BL21, and the recombinant enzyme Cel5G was purified to homogeneity for enzymatic analysis. Cel5G hydrolyzed a wide range of ß-1,4-, ß-1,3/ß-1,4-, or ß-1,3/ß-1,6-linked polysaccharides, amorphous cellulose, filter paper, and microcrystalline cellulose. Its highest activity was in 50 mM citrate buffer, pH 4.8, at 50°C. Cel5G is stable over a wide range of pH values (from 2.0 to 10.6) and is thermally stable under 60°C. It is highly tolerant and active in high salt concentrations and is stable in the presence of pepsin and pancreatin. The K (m) and V (max) values of Cel5G for carboxymethyl cellulose are 19.92 mg/ml and 1,941 µmol min(-1) mg(-1), respectively. These characteristics indicate that Cel5G has potential for industrial use.

[Construction of the shuttle expression-secretion vector with the promoters and signal peptide-encoding sequence from Brevibacillus brevis].

The multiple and tandem promoters and signal peptide-encoding sequence of cell wall protein encoding gene was amplified from Brevibacillus brevis B15 total DNA, the PCR fragment was cloned, sequenced and analyzed, then was submitted to GenBank with a Accession No. AY956423. Another pair of primers were designed to amplify the fragment again, BamHI and Pstl sites was introduced flanking the PCR production. BamHI/Pstl digested fragment was cloned into the corresponding site of shuttle vector pP43NMK to generate the expression-secretion vector pP15MK. The inserted fragment was upstream of mpd gene and the signal peptide-encoding sequence was fused in frame with the mpd gene, which its own signal peptide-encoding sequence was deleted. The recombinant vector was transformed into Bacillus subtilis 1A751, under the control of the promoters and signal peptide from Brevibacillus brevis B15, mpd gene was continuously expressed and secreted at a high efficiency throughout the exponential growth phase and into the late stationary phase, the expression production methyl parathion hydrolase (MPH) was attached on the outside of the cell membrane. MPH activity accumulated to a maximum level of 7.79 U/mL after 48 h of cultivation at the late stationary phase, which was 8.1-fold higher than the expression level of the original Plesiomonas strain M6.

Isolation and characterization of a denitrifying monocrotophos-degrading Paracoccus sp. M-1.

A bacterium strain, which is capable of degrading monocrotophos, was isolated from sludge collected from the bottom of a wastewater treatment system of a chemical factory, and named M-1. On the basis of the results of the cellular morphology, physiological and chemotaxonomic characteristics and phylogenetic similarity of 16S rDNA gene sequences, the strain was identified as a Paracoccus sp. The ability of the strain to mineralize monocrotophos was investigated under different culture conditions. Other organophosphorus insecticides and amide herbicides were also degraded by M-1. The key enzyme (s) involved in the initial biodegradation of monocrotophos in M-1 was shown to be a constitutively expressed cytosolic protein. The addition of M-1 (10(6) CFU g(-1)) to fluvo-aquic soil and a high-sand soil containing monocrotophos (50 mg kg(-1)) resulted in a higher degradation rate than that obtained from noninoculated soil. This microbial culture has great potential utility for the bioremediation of wastewater or soil contaminated with organophosphorus pesticides and amide herbicides.

mazF, a novel counter-selectable marker for unmarked chromosomal manipulation in Bacillus subtilis.

Here, we present a novel method for the directed genetic manipulation of the Bacillus subtilis chromosome free of any selection marker. Our new approach employed the Escherichia coli toxin gene mazF as a counter-selectable marker. The mazF gene was placed under the control of an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible expression system and associated with a spectomycin-resistance gene to form the MazF cassette, which was flanked by two directly-repeated (DR) sequences. A double-crossover event between the linearized delivery vector and the chromosome integrated the MazF cassette into a target locus and yielded an IPTG-sensitive strain with spectomycin-resistance, in which the wild-type chromosome copy had been replaced by the modified copy at the targeted locus. Another single-crossover event between the two DR sequences led to the excision of the MazF cassette and generated a strain with IPTG resistance, thereby realizing the desired alteration to the chromosome without introducing any unwanted selection markers. We used this method repeatedly and successfully to inactivate a specific gene, to introduce a gene of interest and to realize the in-frame deletion of a target gene in the same strain. As there is no prerequisite strain for this method, it will be a powerful and universal tool.

[Recombinant expression and secretion of mpd gene using the promoters and signal peptide-encoding sequences of ytkA and ywoF gene from Bacillus subtilis].

A shuttle promoter-probe vector pNW33N-mpd was constructed with the E. coli-B. subtilis shuttle vector pNW33N and the mature mpd gene without it' s signal peptide-encoding sequence. The promoter fragments of B. subtilis ytkA and ywoF gene were cloned from plasmid pMPDP3 and pMPDP29 then generated the shuttle expression vector pNYTM and pNYWM. Expression vectors pNYTM and pNYWM were transformed into B. subtilis 1A751 to construct the expression strain 1A751 (pNYTM) and 1A751 (pNYTM), in these strains, under the control of the promoters and signal peptides of ytkA and ywoF gene, mpd gene was expressed and secreted with its biological activity; the result showed that the promoter of ytkA gene is much stronger than that of ywoF gene. Then a new shuttle expression-secretion vector pYNMK was constructed using the ytkA gene promoter and the signal peptide-encoding sequence of B. subtilis nprB gene, the expression of mpd gene achieved a higher level using the B. subtilis WB800 as the host, the methyl parathion hydrolase activity accumulated to a maximum level of 10.40 u/mL after 84 h of cultivation at the late stationary phase, which was 10.8-fold higher than the expression level of the original Plesiomonas strain M6, about 91.4% of the recombinant expression production was secreted into the culture medium.

High-level expression and secretion of methyl parathion hydrolase in Bacillus subtilis WB800.

The methyl parathion hydrolase (MPH)-encoding gene mpd was placed under the control of the P43 promoter and Bacillus subtilis nprB signal peptide-encoding sequence. High-level expression and secretion of mature, authentic, and stable MPH were achieved using the protease-deficient strain B. subtilis WB800 as the host.

[Determination of the catalytic structures of methyl parathion hydrolase].

Methyl parathion hydrolase (MPH) is a novel member of organophosphorus hydrolase. In this study, mpd gene was expressed in Escherichia coli DH5alpha with its native promoter. MPH was purified to homogeneity. Results show that metal-chelating compounds cannot inhabit the enzyme activity. Inductively Coupled Plasma-Atomic Emission Spectrometry analysis showed that MPH is a zinc-containing enzyme, the Zinc to enzyme molar ratio is near 2:1. In order to investigate critical residues related to enzymatic activity of MPH, chemical modification reagents EDC, DEPC, butanedione and pyridoxal were tested. Experiment results suggested that aspartate, glutamate, arginine and lysine are not important for enzyme activity. But DEPC, which can modify histidine residue, inactivate the enzyme activity greatly, and the inactivation rate is 9.6 h(-1). This result reflects that histidine residues are essential for enzyme activity. All these results provide basic data for MPH structure and molecular evolution research.

Construction and application of a promoter-trapping vector with methyl parathion hydrolase gene mpd as the reporter.

A facilitative and efficient promoter-trapping vector, pUC-mpd, was constructed with the promoterless methyl parathion hydrolase gene as the reporter. This reporter gene is easily used to clone promoters with different promoting strength on selective plates. Promoter regions of the ytkA and ywoF genes with strong promoting and signal peptide functions were cloned from the Bacillus subtilis 168 genomic promoter library with this vector.