[Identification and denitrification characteristics of an aerobic denitrifier].

An aerobic denitrifier named F1 was isolated from grass fish pond water by BTB culture medium preliminary screening and denitrification activity analysis. The isolate was identified as Pseudomonas stutzeri through morphological feature, biochemical characteristics and 16S rDNA sequence analysis. Further studies showed that the optimal carbon resources for F1 denitrification were sodium acetate, sodium citrate, glucose and sucrose, with which the nitrate removal rate could reach 100%. When C/N ratio was above 10, the nitrogen removal rate of strain F1 was more than 96% and no nitrite was accumulated. The optimum condition for F1 growth and aerobic denitrification was temperature 30 degrees C and pH 7.0. The F1 could tolerate dissolved oxygen level of 3.4-7.2 mg/L, and its nitrogen removal rate was more than 85% in 24 hours. Denitrification process of F1 mainly occurred in the exponential phase with NaNO3 or NaNO2 as nitrogen resource, and its denitrification rate reached 92.51% and 82.73% , respectively after 32 h of culture. These results suggest that strain F1 can denitrify NO3(-) or NO2(-) directly, and can tolerant a high dissolved oxygen level, and these characteristics make it a good candidate for aquaculture water quality treatment.

Effect of the C-terminal domains and terminal residues of catalytic domain on enzymatic activity and thermostability of lichenase from Clostridium thermocellum.

To elucidate the effects of C-terminal domains of LicMB (mature lichenase from Clostridium thermocellum) and terminal residues of LicMB-CD (catalytic domain of LicMB) on the properties of lichenase, a series of truncated genes were constructed and expressed in E. coli. The Thr-Pro box had a positive effect while the dockerin domain had a negative impact on the properties of LicMB. The N-terminal 10-25th and C-terminal 1-9th residues of LicMB-CD were necessary to retain high thermostability while the N-terminal 1-7th and C-terminal 1-3rd residues were not necessary to maintain enzymatic activity.

Nisin-controlled extracellular production of apidaecin in Lactococcus lactis.

Apidaecins are heat-stable, nonhelical antibacterial peptides isolated from lymph fluid of the honeybee (Apis mellifera). These peptides are active against a wide range of gram-negative bacteria and they are the most prominent components of the honeybee humoral defense against microbial invasion. In the present study, one isoform of apidaecin, apidaecin Ho, was expressed extracellularly in the food-grade bacterium Lactococcus lactis. Results showed that expression driven by the lactococcal nisA promoter and Usp45 signal peptide resulted in efficient secretion of apidaecin in L. lactis subsp. cremoris NZ9000. Recombinant apidaecin was purified by gel filtration and semipreparative RP-HPLC, and about 10 mg active recombinant apidaecin was obtained from 1,000 ml culture. This is the first report on the nisin-controlled extracellular production of active apidaecin in L. lacits. The expression and delivery of apidaecin in the food-grade L. lactis may provide a clue to facilitate the widespread application of apidaecin in the control and prevention of gram-negative bacteria infections of human and animals.

Functional and structural characterization of apidaecin and its N-terminal and C-terminal fragments.

Two aspects were studied to elucidate the functional and structural characterization of apidaecin and its N-terminal and C-terminal fragments: (i) Functions of the N-terminal and C-terminal fragments of apidaecin were first studied by measuring their antibacterial activity, their ability to enter Escherichia coli cells and their effects on the activities of beta-galactosidase and alkaline phosphatase. The results indicate that neither the N-terminal nor the C-terminal of apidaecin contains intracellular delivery unit or active segment. (ii) The effect of apidaecin on the ATPase activity of DnaK, and the interactions of apidaecin with E.coli lidless DnaK and DnaK D-E helix were studied. Results showed that apidaecin could interact with the E.coli lidless DnaK protein and stimulate its ATPase activity, but not with E.coli DnaK D-E helix. This indicated that the antimicrobial activity of apidaecin may be shown by stimulating the ATPase activity of DnaK by binding to its conventional substrate-binding site, to decrease its cellular concentration of DnaK by competing with natural substrates and inhibit the enzymes' activities of E. coli cells. It is the first study to suggest that the apidaecin-binding site of DnaK is the conventional substrate binging site.

In vitro assessment of gastrointestinal viability of two photosynthetic bacteria, Rhodopseudomonas palustris and Rhodobacter sphaeroides.

The objectives of this study were to assess the potential of two photosynthetic bacteria (PSB), Rhodopseudomonas palustris HZ0301 and Rhodobacter sphaeroides HZ0302, as probiotics in aquaculture. The viability of HZ0301 and HZ0302 in simulated gastric transit conditions (pH 2.0, pH 3.0 and pH 4.0 gastric juices) and in simulated small intestinal transit conditions (pH 8.0, with or without 0.3% bile salts) was tested. The effects of HZ0301 and HZ0302 on the viability and permeability of intestinal epithelial cell in primary culture of tilapias, Oreochromis nilotica, were also detected. All the treatments were determined with three replicates. The simulated gastric transit tolerance of HZ0301 and HZ0302 strains was pH-dependent and correspondingly showed lower viability at pH 2.0 after 180 min compared with pH 3.0 and pH 4.0. Both HZ0301 and HZ0302 were tolerant to simulated small intestine transit with or without bile salts in our research. Moreover, there was no significant difference (P>0.05) among three treatments including the control and the groups treated with HZ0301 or HZ0302 both in intestinal epithelial cell viability and membrane permeability, showing no cell damage. In summary, this study demonstrated that HZ0301 and HZ0302 had high capacity of upper gastrointestinal transit tolerance and were relatively safe for intestinal epithelial cells of tilapias.

Apidaecin-type peptides: biodiversity, structure-function relationships and mode of action.

Apidaecins (apidaecin-type peptides) refer to a series of small, proline-rich (Pro-rich), 18- to 20-residue peptides produced by insects. They are the largest group of Pro-rich antimicrobial peptides (AMPs) known to date. Structurally, apidaecins consist of two regions, the conserved (constant) region, responsible for the general antibacterial capacity, and the variable region, responsible for the antibacterial spectrum. The small, gene-encoded and unmodified apidaecins are predominantly active against many gram-negative bacteria by special antibacterial mechanisms. The mechanism of action by which apidaecins kill bacteria involves an initial non-specific binding of the peptides to an outer membrane (OM) component. This binding is followed by invasion of the periplasmic space, and by a specific and essentially irreversible combination with a receptor/docking molecule that may be a component of a permease-type transporter system on inner membrane (IM). In the final step, the peptide is translocated into the interior of the cell where it meets its ultimate target. Evidence that apidaecins are non-toxic for human and animal cells is a prerequisite for using them as novel antibiotic drugs. This review presents the biodiversity, structure-function relationships, and mechanism of action of apidaecins.

The nisin-controlled gene expression system: construction, application and improvements.

Lactic acid bacteria are widely used in industrial fermentation. The potential use of these bacteria as homologous and heterologous protein expression hosts has been investigated extensively. The NIsin-Controlled gene Expression system (the NICE system) is an efficient and promising gene expression system based on the autoregulation mechanism of nisin biosynthesis in the Lactococcus lactis. In the NICE system, the membrane-located histidine kinase NisK senses the inducing signal nisin and autophosphorylates, then transfers phosphorous group to intracellular response regulator protein NisR which activates nisA promoter to express the downstream gene(s). The NICE system allows regulated overproduction of a variety of interest proteins by several Gram-positive bacteria, especially L. lactis. The essential elements for system construction, its application for expression of some biotechnologically important proteins and further improvements of this system are discussed.