Fate of transforming bacterial genome following incorporation into competent cells of Bacillus subtilis: a continuous length of incorporated DNA

In contrast to the conventional transformation of Bacillus subtilis using purified DNA, those using DNA in lysed protoplasts have a high transformation efficiency and enable whole-genome transfer into competent B. subtilis [Akamatsu, T. and Taguchi, H., Biosci. Biotechnol. Biochem., 65, 823-829 (2001)]. Here, we examined the length of incorporated continuous DNA by analyzing the cotransfer ratio with selected and unselected markers, on the basis of a new experimental design. The cotransfer ratio of a selected marker with an unselected marker on the opposite side of the genetic map of the B. subtilis chromosome was about 5.6% and could be interpreted as congression (double transformation) ratio. In the wild-type strain, the cotransfer ratio of cysA (113 kb position on 4215 kb of B. subtilis chromosome) with metC (1384 kb) and leuB (2891 kb) was 0.77%, twice the value (5.6% x 5.6%=0.31%) calculated from the congression ratio. Moreover, in a genetic background, the cotransfer ratios of metC with cysA and leuB, and metC with cysA and arg1 (3012 kb) were 2.7% and 7.2%, respectively. These results strongly suggest that the length of continuous DNA incorporated into B. subtilis is most probably greater than 1271 kb. When the DNA from the protoplast lysate was fragmented by mixing, the cotransfer ratios of arg1 with metC, and arg1 with metC and trpC (2374 kb) were 2.8% and 0.16%, respectively. A high cotransfer ratio (2.7-7.2%) could not, therefore, be obtained using the fragmented DNA. Based on these observations, we propose a working hypothesis on the mechanism of the transformation of competent B. subtilis by DNA in protoplast lysates (LP transformation).     

Development of a quantitative real-time PCR method to enumerate total bacterial counts in ready-to-eat fruits and vegetables

A newly developed real-time PCR assay rapidly quantifies the total bacterial numbers in contaminated ready-to-eat vegetables and fruits compared with the standard plate count method. Primers targeting the rpoB gene, which encodes for the beta subunit of the bacterial RNA polymerase and which is common to most bacterial species, was used instead of the 16S rRNA gene, which has multiple copies and varies among bacterial species. A primer pair specific for rpoB was confirmed to amplify rpoB in a wide range of bacterial species after we assessed 49 strains isolated from five kinds of fruits and vegetables. We purchased fruits and vegetables from retail shops and enumerated the bacteria associated with them by use of real-time PCR and compared this to the number found by the culture method. We found a high correlation between the threshold PCR cycle number when compared with the plate count culture number. The real-time PCR assay developed in this study can enumerate the dominant bacterial species in ready-to-eat fruits and vegetables.     

Development of a simple bio-hydrogen production system through dark fermentation by using unique microflora

In order to ensure efficient functioning of hydrogen fermentation systems that use Clostridium as the dominant hydrogen producer, energy-intensive process such as heat pretreatment of inoculum and/or substrate, continuous injection, and control of anaerobic conditions are required. Here, we describe a simple hydrogen fermentation system designed using microflora from leaf-litter cattle-waste compost. Hydrogen and volatile fatty acid production was measured at various hydraulic retention times, and bacterial genera were determined by PCR amplification and sequencing. Although hydrogen fermentation yield was approximately one-third of values reported in previous studies, this system requires no additional treatment and thus may be advantageous in terms of cost and operational control. Interestingly, Clostridium was absent from this system. Instead, Megasphaera elsdenii was the dominant hydrogen-producing bacterium, and lactic acid-producing bacteria (LAB) were prevalent. This study is the first to characterize M. elsdenii as a useful hydrogen producer in hydrogen fermentation systems. These results demonstrate that pretreatment is not necessary for stable hydrogen fermentation using food waste.     

Synthesis and application of fluorescein- and biotin-labeled molecular probes for the chemokine receptor CXCR4

The design, synthesis, and bioevaluation of fluorescence- and biotin-labeled CXCR4 antagonists are described. The modification of D-Lys8 at an epsilon-amino group in the peptide antagonist Ac-TZ14011 derived from polyphemusin II had no significant influence on the potent binding of the peptide to the CXCR4 receptor. The application of the labeled peptides in flow cytometry and confocal microscopy studies demonstrated the selectivity of their binding to the CXCR4 receptor, but not to CXCR7, which was recently reported to be another receptor for stromal cell-derived factor 1 (SDF-1)/CXCL12.     

Syntheses and characterization of xylan esters

A series of low-molecular weight and high-molecular weight xylan esters with different alkyl chain lengths (C2–C12) were synthesized by heterogeneous and homogeneous reactions, respectively. Structure elucidation of these xylan esters was obtained by NMR analysis. The solubility of xylan in CHCl₃ improved after esterification. DSC results did not show a melting peak for all samples. However, WAXD diffractograms show the presence of diffraction peaks. The proposed structural model for xylan ester films is presented. TGA suggested that the thermal stability of xylan increased after esterification. Xylan esters with longer alkyl chains had higher decomposition temperatures. The mechanical properties of the xylan esters were dependent on the alkyl chain length. The tensile strength and elongation at break of these xylan esters ranged from 8 to 29 MPa and from 19 to 44%, respectively. Xylan esters in HFIP can be electrospun into nanofibers.     

Mixed Lubrication with C18 Fatty Acids: Effect of Unsaturation

In this paper, the lubrication mechanism of fatty acids is revisited with a new approach combining experimental and computational chemistry studies. The lubricating properties of single and mixtures of stearic, oleic and linoleic acids in a synthetic Poly-Alpha-Olefin base oil (PAO4) on iron oxide surface are investigated under mixed boundary regime with temperatures from 50 °C up to 150 °C. Low friction coefficient (about 0.055) with no visible wear is reported in the presence of single stearic acid at high temperature. This lubricating behavior is inhibited in the presence of unsaturated fatty acids highlighting an anti-synergic effect of a saturated/unsaturated mixture, especially at 150 °C. To understand the anti-synergic effect and the adsorption mechanism of these molecules, molecular dynamic (MD) and quantum chemistry simulations are performed to evaluate their diffusion coefficient in PAO4 and their adsorption mechanism on iron oxide at different temperatures. MD simulation results show a faster diffusion toward the surface for unsaturated fatty acids than for saturated fatty acid at all the studied temperatures. This means that unsaturated molecules arrive and mainly adsorb before stearic acid on the surface leading to a tribological behavior of the mixture characteristic of the unsaturated molecule. Computational chemistry suggests that all fatty acids (saturated and unsaturated) adsorption mechanism is due to the chemisorption of the carboxylic group on iron oxide surface with no desorption up to 150 °C.     

Detection method of injured Escherichia coli O157 in noodles and vegetables

An enrichment procedure and a polymerase chain reaction (PCR) method for the detection of injured Escherichia coli O157 in foods were examined. Freeze-injured E. coli O157 inoculated in boiled spaghetti could be detected in 6-h culture within 12 h by the PCR method. Cells injured by heating in boiled spaghetti and cells injured by chlorine treatment in raw lettuce and carrot did not grow sufficiently to be detected in 6-h culture but were detected in 18-h culture using selective agar media. The injured cells could be also detected in 18-h culture within 24 h by the PCR method. Enrichment at 42 degrees C in trypticase soy broth (TSB) was more effective than that at 42 degrees C in modified EC broth with novobiocin. These results indicated that the usage of enrichment in TSB for 18 h at 42 degrees C in combination with the PCR method is suitable for screening for E. coli O157 in boiled or chlorinated foods, even if the O157 cells are injured.     

Antibacterial action on pathogenic bacterial spore by green tea catechins

Antibacterial effects of catechins, the major green tea polyphenols, were studied using Clostridium and Bacillus spores. Incubation with crude catechins decreased the number of C botulinum and C butyricum spores but not B cereus spores. Furthermore, the effects of six catechin derivatives on spores were investigated. (−)‐Epicatechin gallate (ECg), (−)‐epigallocatechin (EGC), (−)‐epigallocatechin gallate (EGCg) and (+)‐gallocatechin gallate (GCg) were more effective in decreasing C botulinum and C butyricum spore numbers than (+)‐catechin (C) and (−)‐epicatechin (EC). The vegetative growth of C botulinum and B cereus was inhibited by crude extracts of the catechins. Specifically, purified GCg and EGCg inhibited the vegetative growth of C botulinum and B cereus. The inhibitory effect of ECg on B cereus was similar to that of GCg. However, toxin‐production by B cereus was not inhibited by catechin. Damage to the membrane of C butyricum spores by catechin derivatives was shown using fluorescent microscopy. This study shows that low concentrations of catechins, although requiring a long exposure time, inhibited the growth of bacterial spores. However, the effects of the purified derivatives of the catechins were not the same and GCg and EGCg were found to be the most potent. Spores that are generally resistant to many disinfectants were sensitive to catechins. Copyright © 2005 Society of Chemical Industry