Advances in industrial microbiome based on microbial consortium for biorefinery.

One of the important targets of industrial biotechnology is using cheap biomass resources. The traditional strategy is microbial fermentations with single strain. However, cheap biomass normally contains so complex compositions and impurities that it is very difficult for single microorganism to utilize availably. In order to completely utilize the substrates and produce multiple products in one process, industrial microbiome based on microbial consortium draws more and more attention. In this review, we first briefly described some examples of existing industrial bioprocesses involving microbial consortia. Comparison of 1,3-propanediol production by mixed and pure cultures were then introduced, and interaction relationships between cells in microbial consortium were summarized. Finally, the outlook on how to design and apply microbial consortium in the future was also proposed.

Inhibition of biofilm in Bacillus amyloliquefaciens Q-426 by diketopiperazines.

Biofilm formation can make significant effects on bacteria habits and biological functions. In this study, diketopiperazines (DKPs) produced by strain of Bacillus amyloliquefaciens Q-426 was found to inhibit biofilm formed in the gas-liquid interface. Four kinds of DKPs were extracted from B. amyloliquefaciens Q-426, and we found that 0.04 mg ml(-1) DKPs could obviously inhibit the biofilm formation of the strain. DKPs produced by B. amyloliquefaciens Q-426 made a reduction on extracellular polymeric substance (EPS) components, polysaccharides, proteins, DNAs, etc. Real-time PCR was performed to determine that whether DKPs could make an obvious effect on the expression level for genes related to biofilm formation in the strain. The relative expression level of genes tasA, epsH, epsG and remB which related to proteins, extracellular matrix, and polysaccharides, were downregulated with 0.04 mg ml(-1) DKPs, while the expression level of nuclease gene nuc was significantly upregulated. The quantitative results of the mRNA expression level for these genes concerted with the quantitative results on EPS levels. All of the experimental results ultimately indicated that DKPs could inhibit the biofilm formation of the strain B. amyloliquefaciens Q-426.

The Crystal Structure of the YknZ Extracellular Domain of ABC Transporter YknWXYZ from Bacillus amyloliquefaciens.

Bacillus possesses the peptide toxin Sporulation-Delaying Protein (SDP), which can kill cells within a biofilm to support continued growth, thereby delaying the onset of biofilm sporulation. The four-component transporter YknWXYZ acts as a major SDP efflux pump to protect cells against the endogenous SDP toxin, for which YknYZ is a non-canonical ATP-binding cassette (ABC)-type transporter. YknYZ consists of the following two components: (1) an individual protein (YknY) and (2) a respective permease (YknZ). To date, the crystal structure, molecular function, and mechanism of action of the integral membrane protein YknZ remain to be elucidated. In this study, to characterize the structural and biochemical roles of YknZ in the functional assembly of YknWXYZ, we predicted and overexpressed the YknZ extracellular domain. We determined the crystal structure of B. amyloliquefaciens YknZ at a resolution of 2.0 Å. The structure revealed that the YknZ extracellular region exhibits significant structural similarity with the MacB periplasmic domain, which is a non-canonical ABC-type transporter in the tripartite macrolide-specific efflux pump in Gram-negative bacteria. We also found that the YknZ extracellular domain can directly bind to an extracellular component of YknX. This structural and biochemical study provides insights into the assembly of YknWXYZ, which may be relevant to understanding cannibalistic peptide toxin resistance in Bacillus and controlling bacterial growth.

Crystal structure and functional implications of the tandem-type universal stress protein UspE from Escherichia coli.

BACKGROUND: The universal stress proteins (USP) family member UspE is a tandem-type USP that consists of two Usp domains. The UspE expression levels of the Escherichia coli (E. coli) become elevated in response to oxidative stress and DNA damaging agents, including exposure to mitomycin C, cadmium, and hydrogen peroxide. It has been shown that UspA family members are survival factors during cellular growth arrest. The structures and functions of the UspA family members control the growth of E. coli in animal hosts. While several UspA family members have known structures, the structure of E. coli UspE remains to be elucidated. RESULTS: To understand the biochemical function of UspE, we have determined the crystal structure of E. coli UspE at 3.2 Å resolution. The asymmetric unit contains two protomers related by a non-crystallographic symmetry, and each protomer contains two tandem Usp domains. The crystal structure shows that UspE is folded into a fan-shaped structure similar to that of the tandem-type Usp protein PMI1202 from Proteus mirabilis, and it has a hydrophobic cavity that binds its ligand. Structural analysis revealed that E. coli UspE has two metal ion binding sites, and isothermal titration calorimetry suggested the presence of two Cd(2+) binding sites with a Kd value of 38.3-242.7 µM. Structural analysis suggested that E. coli UspE has two Cd(2+) binding sites (Site I: His117, His 119; Site II: His193, His244). CONCLUSION: The results show that the UspE structure has a hydrophobic pocket. This pocket is strongly bound to an unidentified ligand. Combined with a previous study, the ligand is probably related to an intermediate in lipid A biosynthesis. Subsequently, sequence analysis found that UspE has an ATP binding motif (Gly(269)- X2-Gly(272)-X9-Gly(282)-Asn) in its C-terminal domain, which was confirmed by in vitro ATPase activity monitored using Kinase-Glo® Luminescent Kinase Assay. However, the residues constituting this motif were disordered in the crystal structure, reflecting their intrinsic flexibility. ITC experiments revealed that the UspE probably has two Cd(2+) binding sites. The His117, His 119, His193, and His244 residues within the ß-barrel domain are necessary for Cd(2+) binding to UspE protein. As mentioned above, USPs are associated with several functions, such as cadmium binding, ATPase function, and involvement in lipid A biosynthesis by some unknown way.

Crystallization and preliminary X-ray diffraction analysis of the interaction of Aeromonas hydrophila MtaN-1 with S-adenosylhomocysteine.

Prokaryotic 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MtaN) is a multifunctional enzyme that can hydrolyze S-adenosyl-L-homocysteine (SAH) and S-methyl-5'-thioadenosine (MTA) to give S-ribosyl-L-homocysteine (SRH) and S-methyl-5'-thioribose (MTR), respectively. This reaction plays a key role in several metabolic pathways, including biological methylation, polyamine biosynthesis, methionine recycling and bacterial quorum sensing. Structurally, MtaN belongs to the MtnN subfamily of the purine nucleoside phosphorylase (PNP)/uridine phosphorylase (UDP) phosphorylase family. Aeromonas hydrophila has two MtnN subfamily proteins: MtaN-1, a periplasmic protein with an N-terminal signal sequence, and MtaN-2, a cytosolic protein. In this study, MtaN-1 from Aeromonas hydrophila was successfully expressed and purified using Ni-NTA affinity, Q anion-exchange and gel-filtration chromatography. Crystals of the protein in complex with the substrate SAH were obtained and diffracted to a resolution of 1.4 Å. The crystals belonged to the trigonal space group P3121 or P3221, with unit-cell parameters a = b = 102.7, c = 118.8 Å. The asymmetric unit contained two molecules of MtaN-1 complexed with SAH.

Crystallization and preliminary X-ray diffraction analysis of UspE from Escherichia coli.

Universal stress proteins (Usps) are among the most highly induced genes when bacteria are subjected to several stress conditions such as heat shock, nutrient starvation or the presence of oxidants or other stress agents. Escherichia coli has five small Usps and one tandem-type Usp. UspE (or YdaA) is the tandem-type Usp and consists of two Usp domains arranged in tandem. To date, the structure of UspE remains to be elucidated. To contribute to the molecular understanding of the function of the tandem-type UspE, UspE from E. coli was overexpressed and the recombinant protein was purified using Ni-NTA affinity, Q anion-exchange and gel-filtration chromatography. Crystals of UspE were obtained by sitting-drop vapour diffusion. A diffraction data set was collected to a resolution of 3.2 Šfrom flash-cooled crystals. The crystals belonged to the tetragonal space group I4122 or I4322, with unit-cell parameters a = b = 121.1, c = 241.7 Å.

Determination of diketopiperazines of Burkholderia cepacia CF-66 by gas chromatography-mass spectrometry.

Bacteria communicate with each other by a process termed "quorum sensing" (QS), and diffusible, low-molecular-weight chemicals, called signal molecules, are used as the communication languages. In cell-free Burkholderia cepacia CF-66 culture supernatants, five compounds suspected of being signal molecules were identified. The gene (cepI) related with AHLs synthesis were not detected by polymerase chain reaction (PCR) using specific primers. Gas chromatography-mass spectrometry (GC-MS) revealed that these compounds were not AHLs but the diketopiperazines (DKPs) cyclo(Pro-Phe), cyclo(Pro-Tyr), cyclo(Ala-Val), cyclo(Pro-Leu), and cyclo(Pro-Val), all of which were both D and L-type. Four kinds of DKPs had been isolated from other gram-negative bacteria, but the other was a novel kind discovered in CF-66, and L-cyclo (Pro-Phe) was quantified by GC-MS. It was found that exogenous DKPs had a negative effect on the candidacidal activity of the culture supernatant extracts.

Multiple effects of a novel compound from Burkholderia cepacia against Candida albicans.

A novel compound (named CF66I) produced by Burkholeria cepacia CF-66 strain was investigated for its antifungal activity against Candida albicans. This compound exhibited excellent antifungal activity in a dose- and time-dependent manner. Uptake analysis revealed that the compound preferentially acted against the fungal cell wall, and was also able to enter the cells. Transmission electron microscopy indicated that this compound caused loosening of the cell wall and a significant increase in the cell wall thickness was noted; however, no alterations were observed in the contents of the cell wall components. CF66I probably affected the normal assembly and integration of fungal cell wall components by interrupting the weak interactions between them, such as hydrogen and hydrophobic bonds. Propidium iodide (PI) staining indicated that on exposure to CF66I C. albicans cells became permeable to PI. Marked alterations in lipid and sterol contents were observed, and the major changes were a depletion of total lipids and ergosterol, concomitant with an increase in lanosterol content. These observations suggested that the novel compound CF66I may have considerable potential for development of a new class of antifungal agents.

[Identification and characterization of a Bacillus amyloliquefaciens with high antifungal activity].

Plant disease can cause serious crop losses, and chemical control of disease is costly both to the environment and to the farmer. Some microorganism can produce the substance which has the preventing and exterminating functions to plant pathogens. These substances are valid to plant pathogens with only lower concentration, in addition the substances do not remain in soil and crops without being decomposed. If composization is performed with the microorganism, or the microorganism is mixed into compost, the functional compost having preventing and exterminating action will be made out and that can be more useful to environmental preservation. In order to screen antifungal bacteria for use in biological control, 200 compost samples were taken from different regions in China, over 10 bacterium with clear antifungal activity were isolated from composts, among them, strain Q-12 exhibited the highest antifungal activity which was strongly inhibits the growth of many plant pathogenic fungi such as Fusarium oxysporum and Rhizoctonia solan. According to the characteristics of morphology, physiology and biochemistry tests (API 50 CHB/E system) and the comparison of 16S rDNA sequence, the strain Q-12 was similar to B. subtilis and B. amyloliquefaciens. Some specific genes yyaR, yyaO and tetB, which have previously been shown to be effective for resolving these closely related taxa of the B. subtilis group, were analysed to clarify further the classification of Q-12, and two pairs of primers YyaR _ F/TetB _ R and YyaO _ F/TetB _ R were designed. From the analysis of fingerprints obtained with the two primers, strain Q-12 and B. amyloliquefaciens showed identical genomic fingerprints with primers YyaR _ F/TetB R, indicating their closely genetic relationship, and was identified as B. amyloliquefaciens. In the investigation of the culture condition, growth was carried out in a basal medium and gradually supplemented with the various ingredients to be investigated. The major ingredients being investigated included carbon sources, nitrogen sources and inorganic salts. The optimum medium and culture conditions of the strain all were studied with single factor test. The strain is easy to cultivate, and the optimal antibiotic production condition is growth in a medium (5 g/L glucose, 1 g/L NH4Cl, 0.8 g/L beef extract, 5 g/L MgCl2, pH 6.0) at 33 degrees C for 40 h. A number of Bacillus strains have proven safe over many years as a nonpathogenic species and are consumed in ton quantities in several human food preparations. Strains of Bacillus have several advantages over other biocontrol bacteria in that they are often soil isolates capable of sporulation, easy to cultivate and store, and some strains have been shown to increase yields of various crops.

[Purification and properties of anitibiotic from Burkholderia cepacia CF-66].

Burkholderia cepacia CF-66 isolated from compost showed antifungal activity against plant pathogens such as Rhizoctomia solani and some other fungi. CF66I produced by Burkholderia cepacia CF-66 was separated by gel chromatography using Sephadex-75pg and Sephacryl S-100 column. CF66I has very high degree of stability of antifungal activity under higher temperature and alkaline conditions. Some organic solvents could improve its activity at low concentration. The research for the structure of CF66I find that it is a compound with amide bonds and its main structural units is (CH2CH2O)n.

Purification and properties of a low-molecular-weight phytase from Cladosporium sp. FP-1.

A fungus producing high levels of phytase was isolated from air and identified as Cladosporium sp. The phytase production was stimulated by phytate in the medium used. The maximum production of phytase (108 U/ml) occurred in a medium containing 1.0 g of phytate per 100 ml. The phytase was purified to electrophoretic homogeneity by ion-exchange chromatography and gel filtration. Based on SDS-PAGE analysis, the molecular weight of the purified phytase was calculated to be approximately 32.6 kDa, and the narrow protein band indicated that this phytase is not glycosylated. The phytase has an optimum pH of 3.5, and an optimum temperature of 40 degrees C. The phytase activity was stimulated by 2-mercaptoethanol and dithiothreitol, and inhibited by Ba2+, Pb2+, iodoacetate, p-chloromercuribenzoate and phenylmethylsulfonyl fluoride. The phytase displayed high affinity for phytate and the Km was 15.2+/-3.1 microM. NMR analyses (1D and 2D) indicated that the end hydrolysis product of phytate was myo-inositol 1,2,5-triphosphate.

Purification and properties of a phytase from Candida krusei WZ-001.

A phytase from Candida krusei WZ-001 isolated from soil was purified to electrophoretic homogeneity by ion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration. The phytase is composed of two different subunits with molecular masses of 116 kDa and 31 kDa on SDS-PAGE (or 120 kDa and 30 kDa on gel chromatography), with the larger subunit having a glycosylation rate of around 35%. The phytase has an optimum pH of 4.6, an optimum temperature of 40 degrees C and a pI value of 5.5. The phytase activity was stimulated by 2-mercapto-ethanol and dithiothreitol (DTT), and inhibited by Zn2+, Mg2+, iodoacetate, pI value of 5.5. The phytase activity was stimulated by 2-mercapto-ethanol ethanol and dithiothreitol (DTT), and inhibited by Zn2+, Mg2+, iodoacetate, p-chroloromercuribenzoate (pCMB) and phenylmethylsulfonyl fluoride (PMSF). The phytase displayed a broad substrate specificity and the K(m) for phytate was 0.03 mM. Phytate was sequentially hydrolyzed by the phytase. Furthermore, 1D and 2D NMR analyses and bioassay of myoinositol indicated that the end hydrolysis product of phytate was myoinositol 2-monophosphate.