Bacterial Acclimation Inside an Aqueous Battery.

Specific environmental stresses may lead to induced genomic instability in bacteria, generating beneficial mutants and potentially accelerating the breeding of industrial microorganisms. The environmental stresses inside the aqueous battery may be derived from such conditions as ion shuttle, pH gradient, free radical reaction and electric field. In most industrial and medical applications, electric fields and direct currents are used to kill bacteria and yeast. However, the present study focused on increasing bacterial survival inside an operating battery. Using a bacterial acclimation strategy, both Escherichia coli and Bacillus subtilis were acclimated for 10 battery operation cycles and survived in the battery for over 3 days. The acclimated bacteria changed in cell shape, growth rate and colony color. Further analysis indicated that electrolyte concentration could be one of the major factors determining bacterial survival inside an aqueous battery. The acclimation process significantly improved the viability of both bacteria E. coli and B. subtilis. The viability of acclimated strains was not affected under battery cycle conditions of 0.18-0.80 mA cm(-2) and 1.4-2.1 V. Bacterial addition within 1.0×10(10) cells mL(-1) did not significantly affect battery performance. Because the environmental stress inside the aqueous battery is specific, the use of this battery acclimation strategy may be of great potential for the breeding of industrial microorganisms.

Binding mechanism and electrochemical properties of M13 phage-sulfur composite.

Self-assembly of nanostructured materials has been proven a powerful technique in material design and synthesis. By phage display screening, M13 phage was found to strongly bind sulfur particles. Fourier transform infrared and X-ray photoelectron spectroscopy measurements indicated that the strong sulfur-binding ability of M13 phage derives from newly generated S-O and C-S bonds. Using this phage assembled sulfur composite in a lithium battery, the first discharge capacity reached 1117 mAh g(-1), which is more than twice that of the sulfur only cathode. Besides, the negative polysulfide shuttle effect in a lithium-sulfur battery was significantly suppressed.

A simple and rapid method to isolate purer M13 phage by isoelectric precipitation.

M13 virus (phage) has been extensively used in phage display technology and nanomaterial templating. Our research aimed to use M13 phage to template sulfur nanoparticles for making lithium ion batteries. Traditional methods for harvesting M13 phage from Escherichia coli employ polyethylene glycol (PEG)-based precipitation, and the yield is usually measured by plaque counting. With this method, PEG residue is present in the M13 phage pellet and is difficult to eliminate. To resolve this issue, a method based on isoelectric precipitation was introduced and tested. The isoelectric method resulted in the production of purer phage with a higher yield, compared to the traditional PEG-based method. There is no significant variation in infectivity of the phage prepared using isoelectric precipitation, and the dynamic light scattering data indirectly prove that the phage structure is not damaged by pH adjustment. To maximize phage production, a dry-weight yield curve of M13 phage for various culture times was produced. The yield curve is proportional to the growth curve of E. coli. On a 200-mL culture scale, 0.2 g L(-1) M13 phage (dry-weight) was produced by the isoelectric precipitation method.

Phenazine-1-carboxylic acid derivatives: design, synthesis and biological evaluation against Rhizoctonia solani Kuhn.

Rhizoctonia solani Kuhn is the pathogen that causes sheath blight and results in significant yield reduction in rice and in nearly 50 other crops. In order to develop a new fungicide effective against this pathogen, a series of structurally diverse phenazine-1-carboxylic acid derivatives, 2a, 2b, 2c, 2d, 2e, 2f, 2g, 2h, 2i, 2j, and 2k, were designed, synthesized and evaluated for their antifungal activity. The two most active compounds 2i and 2j were selected as lead compounds for further antifungal research.

Pre-fractionation of rat liver cytosol proteins prior to mass spectrometry-based proteomic analysis using tandem biomimetic affinity chromatography.

Efficient and high resolution separation of the protein mixture prior to trypsin digestion and mass spectrometry (MS) analysis is generally used to reduce the complexity of samples, an approach that highly increases the probability of detecting low-copy-number proteins. Our laboratory has constructed an affinity ligand library composed of thousands of ligands with different protein absorbance effects. Structural differences between these ligands result in different non-bonded protein-ligand interactions, thus each ligand exhibits a specific affinity to some protein groups. In this work, we first selected out several synthetic affinity ligands showing large band distribution differences in proteins absorbance profiles, and a tandem composition of these affinity ligands was used to distribute complex rat liver cytosol into simple subgroups. Ultimately, all the fractions collected from tandem affinity pre-fractionation were digested and then analyzed by LC-MS/MS, which resulted in high confidence identification of 665 unique rat protein groups, 1.8 times as many proteins as were detected in the un-fractionated sample (371 protein groups). Of these, 375 new proteins were identified in tandem fractions, and most of the proteins identified in un-fractionated sample (290, 80%) also emerged in tandem fractions. Most importantly, 430 unique proteins (64.7%) only characterized in specific fractions, indicating that the crude tissue extract was well distributed by tandem affinity fractionation. All detected proteins were bioinformatically annotated according to their physicochemical characteristics (such as MW, pI, GRAVY value, TM Helices). This approach highlighted the sensitivity of this method to a wide variety of protein classes. Combined usage of tandem affinity pre-fractionation with MS-based proteomic analysis is simple, low-cost, and effective, providing the prospect of broad application in proteomics.

Rapid monitoring of mRNA levels with a molecular beacon during microbial fermentation.

In the microbial fermentation bioreactor, the processes of mRNA transcription, protein translation, and enzyme-catalyzed biosynthesis remain as "black boxes" of industrial monitoring and process control. Monitoring the kinetics of these "black boxes" is very helpful for optimizing and controlling the microbial fermentation process. This study first applied a molecular beacon (MB) to monitor the changes in the mRNA level of the phzC gene during antibiotic phenazine-1-carboxylic acid fermentation. Seven typical MB hybridization buffers were compared, and the effect of formamide on MBs was also studied. The results showed that rapid monitoring of the mRNA level using MBs was feasible. The optimal hybridization buffer for phzC MB was 100 mM Tris, 1 mM MgCl(2), pH 8.0. The optimal hybridization temperature was 35 degrees C, and formamide proved unsuitable for MB hybridization. The limit of detection of phzC MB was 1.67 nM and MB hybridization was complete by 7 min. Given that the time for RNA extraction is 12 min, it is possible that monitoring of phzC mRNA can be completed in less than 20 min. Since production of most amine acids, organic acids, wines, antibiotics, and proteins relies on microbial fermentation, our method may have some potential for application in these other microbial industries.

Combined usage of cascade affinity fractionation and LC-MS/MS for the proteomics of adult mouse testis.

In this report, the proteomics of adult mouse testis were analyzed by the combined usage of cascade affinity fractionation and LC-MS/MS. The differences between the selected affinity ligands in size, shape, structure, and biochemical characteristics, result in each ligand exhibiting a specific affinity to some protein groups. Therefore, a cascade composition of different ligands can be applied to the fractionation of complex tissue proteins. Ultimately, the fractions collected from cascade affinity fractionation were analyzed by LC-MS/MS, which resulted in high confidence identification of a total of 1378 non-redundant mouse testis protein groups, over 2.6 times as many proteins as were detected in the un-fractionated sample (526). All detected proteins were bioinformatically categorized according to their physicochemical characteristics (such as relative molecular mass, pI, grand average hydrophobicity value, and transmembrane helices), subcellular location, and function annotation. This approach highlighted the sensitivity of this method to a wide variety of protein classes. Utilizing a combination of cascade affinity fractionation and LC-MS/MS, we have established the largest proteomic database for adult mouse testis at the present time.

A novel fractionation method prior to MS-based proteomics analysis using cascade biomimetic affinity chromatography.

This is the first report that combines cascade biomimetic affinity fractionation with MS-based proteomics analysis. Our lab has constructed an affinity ligand library composed of thousands of ligands with different protein-binding properties. Structural differences between these ligands result in different non-bonded protein-ligand interactions, thus each ligand exhibits a specific affinity to some protein groups. In this work, we first screened out three affinity ligands with large difference in protein-binding properties. Next, cascade combination of these ligands was applied to fractionate tissue sample into simple subgroups prior to trypsin digestion and LC-MS/MS analysis. In this study, 391 non-redundant protein groups were identified in unfractionated rat liver cytosol, 499 protein groups were identified in 2 fractions of the first affinity fractionation, 616 in 4 fractions of the second fractionation, and 738 in 8 fractions of the third fractionation (an 88.74% increase). Ultimately, a total of 859 unique protein groups were identified in all cascade fractions (a 119.6% increase compared with unfractionated sample). The proteins detected in each fraction were bioinformatically categorized according to their physicochemical characteristics (relative molecular mass, pI, GRAVY value and TM helices). This approach highlighted the sensitivity of this method to a wide variety of protein classes.

Utilizing RNA/DNA hybridization to directly quantify mRNA levels in microbial fermentation samples.

mRNA quantification has become a research hotspot. Quantitative real-time RT-PCR is a popular method but is known to lack precision. To rapidly monitor the kinetics of mRNA levels for the control of microbial fermentation processes, we developed an SYBR Green I-based universal method to directly quantify mRNA from fermentation samples. After total RNA was extracted, the mRNA was hybridized and protected by a longer DNA oligonucleotide. The probe length determined the strength of signal amplification. S1 nuclease and RNase A were used to remove excess probe, single-stranded RNA, and mis-matched RNA/DNA hybrids. Finally, the perfect-matched RNA/DNA hybrid was quantified by SYBR Green I dye. The conditions of liquid hybridization and enzyme digestion were systemically optimized. The kinetic tendency of phzC mRNA levels during phenazine-1-carboxylic acid fermentation was consistent with the results from MB hybridization in our previous report. The detection of mRNA levels of ten genes in Pseudomonas sp. M18G proved that this method is universal and feasible for mRNA quantification, and has great potential for application in mRNA quantification in various organisms.

Comparative analysis of depurination catalyzed by ricin A-chain on synthetic 32mer and 25mer oligoribonucleotides mimicking the sarcin/ricin domain of the rat 28S rRNA and E. coli 23S rRNA.

Ricin A-chain can inactivate eukaryotic ribosomes, but exhibits no N-glycosidase activity on intact E. coli ribosomes. In the present research, in order to avoid using radiolabeled oligoribonucleotides, two kinds of synthetic 5'-FAM fluorescence-labeled oligoribonucleotide substrates were used to mimic the sarcin/ricin domains of rat 28S rRNA and E. coli 23S rRNA (32mer and 25mer, named as Rat FAM-SRD and E. coli FAM-SRD, respectively). Ricin A-chain was able to specifically release adenine from the first adenosine of the GAGA tetraloop and exhibited specific N-glycosidase activity under neutral and weak acidic conditions with both substrates. However, under more acidic conditions, ricin A-chain was able to release purines from other sites on eukaryotic substrates, but it retained specific depurination activity on prokaryotic substrates. At pH 5.0, the Michaelis constant (K(m)) for the reaction with Rat FAM-SRD (4.57+/-0.28microM) corresponded to that with E. coli FAM-SRD (4.64+/-0.26microM). However, the maximum velocity (V(max)) for ricin A-chain with Rat FAM-SRD was 0.5+/-0.024microM/min, which is higher than that with E. coli FAM-SRD (0.32+/-0.011microM/min).

Structural and biological characterization of a novel acutobin-like enzyme isolated from the venom of the sharp-nosed pit viper (Deinagkistrodon acutus).

We previously reported the purification of a serine proteinase from the venom of the sharp-nosed pit viper (Deinagkistrodon acutus) using a combination of affinity chromatography and ion-exchange chromatography [Xin, Dong, Wang and Li, R. (2007) J. Chromatogr. B 859, 111-118]. The high fibrinogen-clotting activity [2025 NIH (National Institutes of Health) units/mg] of this protein indicated that it may have great potential as a drug for treating thrombolysis. In order to systemically determine the purified protein's structure and activity, it was characterized using the following methods: MS, isoelectric focusing, deglycosylation analysis, amino acid composition analysis, peptide mass fingerprinting, N-terminal amino acid sequencing, CD, hydrophobic-site analysis and bioactivity assays. In addition, a fluorescence probe was synthesized and conjugated to the protein in order to analyse its active site. The results indicated that the protein is a novel acutobin-like enzyme (designated acutobin II) with strong clotting and esterase activities and is composed of a 28 kDa peptide chain plus approx. 6 kDa of O-linked glycan chains. The protein contains 249 amino acids and, remarkably, no tryptophan residues. The pI of the protein is 4.8+/-0.2. The protein's secondary structure is dominated by beta-sheets (49%) and random coils (43%), and its tertiary structure does not contain any metal ions or disulfide bonds and possesses only one hydrophobic pocket. Analysis revealed that the hydrophobic pocket is most likely the enzymatic active site.

An easily-automated assay for the physiological state quantification of Pseudomonas sp. M18.

In order to foreknow poorly performing cultures before wasting energy to scale them to large cultures, industrial microbial fermentation can greatly benefit from knowledge of the physiological state of cells. The method currently proposed is an easily automated physiological state determination method. We have designed one universal rRNA-specific probe for bacteria and developed novel signal probe hybridization (SPH) assay featuring no RNA extraction and no PCR amplification steps necessary to quantify the physiological state of microbial cells. The microbial cell was lysed with sonication and SDS. Signal probes were applied to hybridize and protect the rRNA target. S1 nuclease was then applied to remove the excessive signal probes, the single-stranded RNA and the mismatch RNA/DNA hybrids. The remaining signal probe was captured with a corresponding capture probe immobilized on a microplate and quantified with a horseradish peroxidase-conjugated color reaction. We then systemically optimized our assay. Results showed that the cell limit of detection (LOD) and the cell limit of quantification (LOQ) were 2.64 x 10(4) cells and 9.86 x 10(4) cells per well of microplate, respectively. The limit of detection (LOD) and the limit of quantification (LOQ) of signal probe were 49.0 fM and 344.0 fM respectively. Using this technique, we quantified the 16S rRNA levels during the fermentation process of Pseudomonas sp. M18. Our results indicate that the 16S rRNA levels can directly inform us about the physiological state of microbial cells. This technique has great potential for application to the microbial fermentation industry.

Affinity purification of egg yolk immunoglobulins (IgY) with a stable synthetic ligand.

Chicken IgY (egg yolk immunoglobulin) is a functional equivalent of mammalian IgG. Traditional methods for IgY purification involve multi-step procedures that result in low recovery of IgY. After a large scale screening of our 700-member synthetic ligand library synthesized by epichlorohydrin and cyanuric chloride methods, a high efficiency ligand of IgY was found. By one-step purification with this ligand, the purity of IgY could reach 92.1%, and the recovery of IgY could reach 78.2%. This synthetic ligand had a higher binding capacity of 74.8 mg IgY/ml and had no negative effects on immunoreactivity. Remarkably, this ligand was also highly stable and could resist 1M NaOH, thus having great potential for the industrial-scale production of IgY.

Utilizing a library of synthetic affinity ligands for the enrichment, depletion and one-step purification of leech proteins.

Although the concept of affinity purification using synthetic ligands had been utilized for many years, there are few articles related to this research area, and they focus only on the affinity purification of specific protein by a defined library of synthetic ligands. This study presents the design and construction of a 700-member library of synthetic ligands in detail. We selected 297 ligand columns from a 700-member library of synthetic ligands to screen leech protein extract. Of the 297, 154 columns had an enrichment effect, 83 columns had a depletion effect, 36 columns had a one-step purification effect, and 58 columns had a one-step purification via flowthrough effect. The experimental results achieved by this large library of affinity ligands provide solid convincing data for the theory that affinity chromatography could be used for the enrichment of proteins that are present in low abundance, the depletion of high abundance proteins, and one-step purification of special proteins.

Biomimetic affinity purification of cardiotoxin and its pharmacological effects on the nervous system.

Cobra venom is a very precious natural resource. The traditional method for purification of cardiotoxin from cobra venom is a multi-step, high cost, and low recovery procedure. By molecular modeling and docking with SYBYL software, we designed and synthesized an affinity ligand, m-aminobenzoic acid, for high efficiency purification of this therapeutically useful Chinese cobra venom cardiotoxin. The one-step recovery of cardiotoxin reached 64% and the purity reached 92% upon purification. The binding capacity of this synthetic ligand was 9.1 mg cardiotoxin/g moist weight gel and the affinity constant for cardiotoxin was 5.5 x 10(3) M(-1). Unlike a natural affinity ligand, this synthetic ligand is highly stable, and has great potential for industrial scale production of cardiotoxin. In addition, we examined the effects of cardiotoxin on the nervous system in a mouse model. Results showed that cardiotoxin could maintain analgesic effects for 120 min with a dose of less than 0.06 mg/kg (2.8% of the LD(50)). Administration of 0.12 mg/kg cardiotoxin could improve scopolamine impairments of memory in mice. These results suggest that cardiotoxin may be a potential drug for nervous system diseases.

Affinity purification of serine proteinase from Deinagkistrodon acutus venom.

An affinity protocol was developed for the preparation of the main serine proteinase from Deinagkistrodon acutus venom on industrial scales. As affinity ligand, l-arginine was composed to medium and its structure was confirmed by ESI-MS analysis. The purification process consisted of one major affinity chromatography step to remove more than 95% of other proteins, and a polishing step of DEAE ion-exchange chromatography for removal of minor contaminants. The serine proteinase was 100% pure analyzed on HPLC Vydac C4 column, 99.4% on TSK G3000SW column, and 97.7% with SDS-PAGE analysis. The yield of the main serine proteinase was 3.6% of crude venom protein, the recoveries of typical fibrinogen (Fg) clotting activity and arginine esterase activity of serine proteinase were 82.2% and 84%, higher than those of other reported traditional protocols, the proteinase also showed arginine amidase activity. Reducing SDS-PAGE analysis showed that the arginine esterase was a single polypeptide with the mass of approximately 40 kDa, while MALDI-TOF-TOF-MS analysis showed that the purified proteinase should be a approximately 34 kDa glycoprotein. The desorption constant Kd and the theoretical maximum absorption Qmax on the affinity medium were 9.93 x 10(-5) and 38.1mg/g medium in absorption analysis.

An rhl-like quorum-sensing system negatively regulates pyoluteorin production in Pseudomonas sp. M18.

Pseudomonas sp. M18, isolated from the watermelon rhizosphere, is antagonistic against a number of soil-borne pathogens. This strain produces an uncharacterized red pigment, pyoluteorin (Plt), and two N-acylhomoserine lactones (AHLs). A previously isolated red-pigment-defective mutant, M18-T510, contains an insert within a gene similar to rhlI in P. aeruginosa PAO1. The M18 rhlI gene product is responsible for the production of two AHL signals: N-butyryl-homoserine lactone and N-hexanoylhomoserine lactone. Mutants defective in either rhlI or rhlR showed enhanced Plt biosynthesis due to loss of transcriptional repression, which was mediated, at least in part, by suppressed expression of the activator PltR. A Plt-specific ABC transporter was also upregulated in the rhl mutants in a Plt-dependent manner. In comparison with the wild-type strain, the rhl mutants survived longer during stationary-phase growth.

Immediate oxidative burst onto the leaves after cutting the stem or flooding the roots of Nicotiana benthamiana.

Plants may use a common defense system to respond to various abiotic and biotic stresses. Some hypotheses have suggested that there is a systemic signal that originates from the site of stress and that translocates to distant parts of the plant. We have detected an immediate oxidative burst onto the leaves after cutting the stem that lasted for 3 minutes and an immediate oxidative burst onto the leaves after flooding the roots of Nicotiana benthamiana that lasted for 20 minutes. Both of the two oxidative bursts were generated within 30 seconds. This strongly indicated that the systemic signal was not a transportable molecule from the site of stress and that it could be some form of electrical signalling.

Genetic diversity of phenazine- and pyoluteorin-producing pseudomonads isolated from green pepper rhizosphere.

The genetic diversity among indigenous phenazine-1-carboxylic acid (PCA)-producing and pyoluteorin (Plt)-producing isolates of pseudomonads screened from green pepper rhizosphere was exploited in this study. A total of 48 bacterium isolates producing one or both of these antibiotics were screened from green pepper rhizosphere in diverse regions in China. Among these isolates, 45 could produce PCA, 3 could produce both PCA and Plt, and none could produce Plt only. Based on the restriction patterns of partial 16S and 16S-23S internal transcribed spacer (ITS) PCR fragments generated by enzyme HaeIII or HinfI, these isolates fell into 19 or 17 distinct groups respectively, indicating that there was a significant diversity among them. Polygenetic analysis of the partial 16S rDNA and 16S-23S ITS sequence from the representative in each group in the context of similar sequence from previously described bacterial species indicated that most isolates were closely related to the species of Pseudomonas fluorescens, P. putida, and Stenotrophomonas maltophilia. Some of these representatives of these isolates, then, are likely to be novel strains or species in these two genera.

Removal of humic substances from soil DNA using aluminium sulfate.

Direct extraction of soil DNA has become essential for the study of soil microorganisms. Humic substances co-extracted during DNA retrieval is a big problem because it greatly inhibits the enzymes involved in manipulating DNA. Popular commercial kits available for soil DNA extraction are unable to overcome this problem. Here we report an effective protocol for the removal of humic substance from soil DNA. The protocol involves flocculation of the humic substance by excessive Al(3+), then removal of superfluous Al(3+) via pH adjustment and finally release of soil microbial DNA by SDS lysis. This technique is superior to that employed by the UltraClean Soil DNA Kit and can be applied to a wide variety of soils.