Reactance and resistance: main properties to follow the cell differentiation process in Bacillus thuringiensis by dielectric spectroscopy in real time.

During growth, Bacillus thuringiensis presents three phases: exponential phase (EP), transition state (TS), and sporulation phase (SP). In order to form a dormant spore and to synthesize delta-endotoxins during SP, bacteria must undergo a cellular differentiation process initiated during the TS. Dielectric spectroscopy is a technique that can be utilized for continuous and in situ monitoring of the cellular state. In order to study on-line cell behavior in B. thuringiensis cultures, we conducted a number of batch cultures under different conditions, by scanning 200 frequencies from 42 Hz to 5 MHz and applying fixed current and voltage of 20 mA and 5 V DC, respectively. The resulting signals included Impedance (Z), Angle phase (Deg), Voltage (V), Current (I), Conductance (G), Reactance (X), and Resistance (R). Individual raw data relating to observed dielectric property profiles were correlated with the different growth phases established using data from cellular growth, cry1Ac gene expression, and free spores obtained with conventional techniques and fermentation parameters. Based on these correlations, frequencies of 0.1, 0.5, and 1.225 MHz were selected for the purpose of measuring dielectric properties in independent batch cultures, at a fixed frequency. X and R manifest more propitious behavior in relation to EP, TS, SP, and spore release, due to particular changes in their signals. Interestingly, these profiles underwent pronounced changes during EP and TS that were not noticed when using conventional methods, but were indicative of the beginning of the B. thuringiensis cell differentiation process.

Extracellular expression of glucose inhibition-resistant Cellulomonas flavigena PN-120 ß-glucosidase by a diploid strain of Saccharomyces cerevisiae.

The catalytic fraction of the Cellulomonas flavigena PN-120 oligomeric ß-glucosidase (BGLA) was expressed both intra- and extracellularly in a recombinant diploid of Saccharomyces cerevisiae, under limited nutrient conditions. The recombinant enzyme (BGLA¹5) expressed in the supernatant of a rich medium showed 582 IU/L and 99.4 IU/g dry cell, with p-nitrophenyl-ß-D-glucopyranoside as substrate. BGLA¹5 displayed activity against cello-oligosaccharides with 2-5 glucose monomers, demonstrating that the protein is not specific for cellobiose and that the oligomeric structure is not essential for ß-D-1,4-bond hydrolysis. Native ß-glucosidase is inhibited almost completely at 160 mM glucose, thus limiting cellobiose hydrolysis. At 200 mM glucose concentration, BGLA¹5 retained more than 50 % of its maximal activity, and even at 500 mM glucose concentration, more than 30 % of its activity was preserved. Due to these characteristics of BGLA¹5 activity, recombinant S. cerevisiae is able to utilize cellulosic materials (cello-oligosaccharides) to produce bioethanol.

Production of cellulases and xylanases under catabolic repression conditions from mutant PR-22 of Cellulomonas flavigena.

Derepressed mutant PR-22 was obtained by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenic treatment of Cellulomonas flavigena PN-120. This mutant improved its xylanolytic activity from 26.9 to 40 U mg(-1) and cellulolytic activity from 1.9 to 4 U mg(-1); this represented rates almost 2 and 1.5 times higher, respectively, compared to its parent strain growing in sugarcane bagasse. Either glucose or cellobiose was added to cultures of C. flavigena PN-120 and mutant PR-22 induced with sugarcane bagasse in batch culture. The inhibitory effect of glucose on xylanase activity was more noticeable for parent strain PN-120 than for mutant PR-22. When 20 mM glucose was added, the xylanolytic activity decreased 41% compared to the culture grown without glucose in mutant PR-22, whereas in the PN-120 strain the xylanolytic activity decreased by 49% at the same conditions compared to its own control. Addition of 10 and 15 mM of glucose did not adversely affect CMCase activity in PR-22, but glucose at 20 mM inhibited the enzymatic activity by 28%. The CMCase activity of the PN-120 strain was more sensitive to glucose than PR-22, with a reduction of CMCase activity in the range of 20-32%. Cellobiose had a more significant effect on xylanase and CMCase activities than glucose did in the mutant PR-22 and parent strain. Nevertheless, the activities under both conditions were always higher in the mutant PR-22 than in the PN-120 strain. Enzymatic saccharification experiments showed that it is possible to accumulate up to 10 g l(-1) of total soluble sugars from pretreated sugarcane bagasse with the concentrated enzymatic crude extract from mutant PR-22.

Light-regulated asexual reproduction in Paecilomyces fumosoroseus.

The entomopathogenic fungus Paecilomyces fumosoroseus has been successfully used in the control of several insect pests. Asexually produced spores (conidia) are the means for dispersal and transmission of the entomopathogen; upon contact with the insect cuticle they germinate and penetrate the host. In model fungal systems it has been found that phototropism, resetting of the circadian rhythm, the induction of carotenogenesis and the development of reproductive structures are controlled by blue light. The effect of light quality on conidial yield of P. fumosoroseus was investigated. Incubation in total darkness resulted in continued vegetative growth and lack of reproductive structures. In contrast, growth of the fungus in continuous illumination or under a night-day regime resulted in prolific formation of conidiophores bearing abundant mature conidia. Conidiation was photoinduced in competent mycelia by a single pulse of blue light and colonies were competent only after they had grown at least 72 h under total darkness. The fluence-response curves generated with blue light indicated that the minimal fluence required for the photomorphogenetic response was 180 micro mol m(-2) and the half-maximal response was at 400 micro mol m(-2). A fluence of 540 micro mol m(-2) was enough to saturate the system, inducing the maximum production of 2.12x10(8) conidia per colony. Higher light intensities markedly decreased conidiation, suggesting the occurrence of a process of adaptation. The authors propose the existence of a dual light-perception system with at least two photoreceptors in P. fumosoroseus, one promoting and one inhibiting conidiation.