Monitoring growth and movement of Ralstonia solanacearum cells harboring plasmid pRSS12 derived from bacteriophage ϕRSS1

We monitored growth and movement of Ralstonia solanacearum harboring the plasmid pRSS12 in tomato seedlings. The plasmid contains a gene for green fluorescent protein (GFP) and is stably maintained in R. solanacearum cells without selection pressure. Bacteria harboring the plasmid can be tracked in planta by visualizing GFP fluorescence. Stems of seedlings were infected with R. solanacearum cells transformed with pRSS12, and bacterial growth and movement, particularly around the vascular bundles, were monitored for more than 7 days. Our results showed that vascular bundles are independent of each other within the stem, and that it takes a long time for R. solanacearum cells to migrate from one vascular bundle to another. For real-time monitoring of bacteria in planta, tomato seedlings were grown on agar medium and bacterial suspension was applied to the root apex. The bacterial invasion process was monitored by fluorescent microscopy. Bacteria invaded taproots within 6 h, and movement of the bacteria was observed until 144 h after inoculation. In susceptible tomato cultivars, strong GFP fluorescence was observed in hypocotyls and lateral roots as well as the taproot. In resistant cultivars, however, GFP fluorescence was rarely observed on lateral roots. Our results show that this monitoring system can be used to assess bacterial pathogenicity efficiently.     

Convenient transformation of anamorphic basidiomycetous yeasts belonging to genus pseudozyma induced by electroporation

A convenient procedure for carrying out transformation by electroporation was optimized for the genus Pseudozyma. Successful transformation was achieved using a plasmid, pUXV1, that confers resistance to hygromycin B; the maximum transformation efficiency was 48 transformants/mug of plasmid DNA. Transformants of Pseudozyma antarctica T-34 expressing a green fluorescent protein were obtained by the procedure.     

Generation of a green fluorescent protein gene chromosomal insertion containing Escherichia coli strain for gene induction-based quantification of bioavailable lysine

The importance of lysine determination in feed materials is crucial for the feed industry because this amino acid can be limiting in many of the cereal materials used for animal feeds. The bacterial gene induction-based assay developed in this study aimed to measure lysine bioavailability in feeds as an alternative analytical method for animal assays. The advantages of a gene induction-based approach include rapid and quantitative estimation of many samples and results that relate a bacterial response to a biological response observed in animals. A whole-cell biosensor strain was constructed using a fluorescent E. coli strain that has an inducible fluorescent phenotype sensitive to extracellular lysine contents. A genetic fusion that links the promoter of cad operon with a green fluorescent protein encoding gene (gfp) was constructed, and a fluorescent assay was developed. A standard lysine curve (R² = 0.95) was generated and used for lysine bioavailability quantification of four feed ingredients (whole egg protein, blood-, soybean-, and meat and bone meal). Quantities as low as 50 μg/ml protein of digested samples were sufficient for analyses using the biosensor, except for meat and bone meal. Because of the low levels of free lysine in non-digested samples, fluorescence of these protein sources containing lower than 500 μg/ml protein was not detected (except for soybean meal). The results using enzymatically digested protein sources showed that the test strain emitted a fluorescent response that was proportional to the level of lysine present in the feed samples.     

Characterization of two 3-hydroxybutyrate dehydrogenases in poly(3-hydroxybutyrate)-degradable bacterium, Ralstonia pickettii T1

Two D-(-)-3-hydroxybutyrate (3HB) dehydrogenases, BDH1 and BDH2, were isolated and purified from a poly(3-hydroxybutyrate) (PHB)-degradable bacterium, Ralstonia pickettii T1. BDH1 activity increased in R. pickettii T1 cells grown on several organic acids as a carbon source but not on 3HB, whereas BDH2 activity markedly increased in the same cells grown on 3HB or PHB. To examine their biochemical properties, bdh1 and bdh2 were cloned and overexpressed in Escherichia coli, and their purified products were characterized. The kinetic parameters indicate that BDH1 is more suitable for converting acetoacetate to 3HB than BDH2, whereas BDH2 is more efficient for the reverse reaction than BDH1. Thus, R. pickettii T1 contains two BDHs with different biochemical properties and physiological roles: BDH1 for cell growth on organic acids other than 3HB and BDH2 for cell growth on 3HB or PHB.     

Fermentative production of (R)-(-)-3-hydroxybutyrate using 3-hydroxybutyrate dehydrogenase null mutant of Ralstonia eutropha and recombinant Escherichia coli

Two systems, one using an (R)-(-)-3-hydroxybutyrate dehydrogenase (BDH) null mutant of Ralstonia eutropha and the other using a recombinant Escherichia coli strain containing a synthetic poly[(R)-(-)-3-hydroxybutyrate] (PHB) operon and an extracellular PHB depolymerase gene, were used for the fermentative production of (R)-(-)-3-hydroxybutyrate (3HB). The concentration of 3HB in the culture supernatant of the mutant R. eutropha system reached about 30 mM after 5 d under anaerobic conditions, although it was about 4-10 mM under aerobic conditions. On the other hand, the 3HB concentration in the culture supernatant of the recombinant E. coli system reached about 70 mM after 4 d, indicating that about 70% of the glucose added was converted to 3HB.     

Cloning of inulin fructotransferase (DFA III-producing) gene from Arthrobacter globiformis C11-1

A gene encoding an inulin fructotransferase (DFA III-producing) [EC 2.4.1.93] from Arthrobacter globiformis C11-1 was cloned and the nucleotide sequence was determined. The cloned fragment contained a 1353 bp open reading frame. The initiation codon was estimated to be an unusual codon, GTG. The gene encoded a signal peptide (40 amino acid residues) for secretion. The molecular mass of the native enzyme was calculated as 43,400 Da from the sequencing data. The deduced amino acid sequence of the enzyme had 74.0 % homology with that of inulin fructotransferase (DFA III-producing) from Arthrobacter sp. H65-7. It also had 45.1% homology with that of inulin fructotransferase (DFA I-producing) [EC 2.4.1.200] from Arthrobacter globiformis S14-3. The enzyme produced in the culture supernatant of an Escherichia coli clone was purified to the electrophoretically homogeneous stage. The N-terminal amino acid sequence of the cloned enzyme secreted in the broth was the same as that of the native enzyme from A. globiformis C11-1. Therefore, on this enzyme, it is estimated that the cleavage sites by the signal peptidase for secretion of A. globiformis C11-1 and E. coli JM109 are the same.     

Analyses of diverse mammals’ milk and lactoferrin glycans using five pathogenic bacterial lectins

Milk indigested glycans hamper infections by blocking pathogen adhesion to babies’ cells via lectins (sugar-binding proteins). This study describes usage of five pathogenic bacterial lectins and two plant lectins for analyses of alpaca, buffalo, camel, cow, dog, fallow deer, giraffe, goat, horse, human, rabbit, and sheep milks, and also commercial human and cow milk lactoferrins. The lectins used differentially reacted with the 12 milks – most strongly with humans’ ones. Most of them (excluding Pseudomonas aeruginosa galactophilic PA-IL) were also sensitive to the human and cow lactoferrins. The fucophilic bacterial lectins were most sensitive to human lactoferrin, while the mannophilic ones – to the cow’s. The actual function of bacterial lectins in pathogen adhesion and their non-glycosylated structures (evading non-specific interactions) are advantageous for such studies. This study shows the efficiency of the bacterial lectins for milk analyses: differentiating between the diverse milks, estimating their anti-infection potentials, and probing their active glycans.     

Characterisation of 2-Cys peroxiredoxin isozyme (Prx1) from Taiwanofungus camphorata (Niu-chang-chih): Expression and enzyme properties

Peroxiredoxins (Prxs) are a family of antioxidant peroxidases. The functions of Prxs comprises of cell protection against oxidative stress and regulation of cell proliferation. A putative 2-Cys Prx isozyme (Prx1) cDNA was cloned from Taiwanofungus camphorata (commonly known as Niu-chang-chih in Taiwan). The deduced amino acid sequence is conserved amongst the reported Prxs. A 3-D homology structure was created for this Prx1. To characterise the T. camphorata Prx1, the coding region was subcloned into a pAVD10 and transformed into Escherichia coli. The recombinant 6His-tagged Prx1 was expressed and purified by Ni²⁺-nitrilotriacetic acid sepharose. The purified enzyme showed two forms using a 15% SDS–PAGE. The enzyme retained 60% activity at 60 °C for 2.5 min. The enzyme was stable under a broad pH range from 5 to 11. The enzyme showed 57% activity after 40 min of incubation at 37 °C with trypsin. The ability of the enzyme to protect intact supercoiled plasmid DNA from ^·OH induced nicking was demonstrated.     

Antioxidant activities of phlorotannins purified from Ecklonia cava on free radical scavenging using ESR and H2O2-mediated DNA damage

The potential antioxidant activities of three phlorotannins (phloroglucinol, eckol and dieckol) purified from Ecklonia cava collected in Jeju Island were investigated to evaluate their potential value as the natural products for foods or cosmetic application. In this study, antioxidant activities were measured by electron spin resonance spectrometry (ESR) technique for scavenging effects of free radicals such as 1,1-diphenyl-2-picrylhydrazyl (DPPH), alkyl, hydroxyl (HO^•) and superoxide anion radical (O₂ ^•−) and by comet assay for protecting effects against H₂O₂-mediated DNA damage. The results show that all the phlorotannins have the potential DPPH, alkyl, hydroxyl and superoxide radical scavenging activities. Especially, eckol samples scavenged around 93% of DPPH at 0.25, 0.5, 1 mg/mL of concentrations and were higher than the other phlorotannins, such as phloroglucinol and dieckol samples. Also, protecting effects of the phlorotannins against H₂O₂-mediated DNA damage increased with increased concentrations of the samples in the L5178 mouse T-cell lymphoma cell lines (L5178Y-R). In conclusion, these results suggest that the three phlorotannins purified from E. cava have the potential inhibitory effect on H₂O₂-mediated DNA damage and harmful free radicals and can be used as antioxidants in cosmetic, foods and drug industry.     

Triggering mechanism of L-glutamate overproduction by DtsR1 in coryneform bacteria

The mechanism of L-glutamate-overproduction by Corynebacterium glutamicum, a biotin auxotroph, is very unique and interesting. L-Glutamate overproduction by this bacterium is induced by biotin-limitation and suppressed by an excess of biotin. Addition of a surfactant or penicillin is also induces L-glutamate overproduction even under excess biotin. After the development of general molecular biological tools such as cloning vectors and DNA transfer techniques, genes encoding biosynthetic enzymes were isolated. With those genes and tools, recombinant DNA technology can be applied to the analysis of biosynthetic pathways and the construction of C. glutamicum strains. In this review, recent studies on the triggering mechanism of L-glutamate overproduction by C. glutamicum are discussed. Disruption of the dtsR1 gene, which encodes a putative component of a biotin-containing enzyme complex that is involved in fatty acid synthesis, causes constitutive overproduction of L-glutamate. As in the case of biotin-limitation, i.e., addition of a surfactant or penicillin, dtsR1-disruption also reduces the activity of the 2-oxoglutarate dehydrogense complex (ODHC). These results indicate that the DtsR1 level affects the activity of ODHC. In our recent studies, a novel regulatory factor that suppresses the expression of DtsR1 was determined. Based on these findings, the triggering mechanism of L-glutamate overproduction is expected to be clarified in more detail.