The cellular location and effect on nisin immunity of the NisI protein from Lactococcus lactis N8 expressed in Escherichia coli and L. lactis

Abstract Vibrio anguillarum and Pasteurella piscicida are Gram-negative bacteria which are pathogenic for marine fish and we report here the first successful transformation of these two bacteria by electroporation. The optimal conditions for electroporation included a field strength of 12.5 kV cm^t-1 and a time constant of 5 ms using 0.2-cm cuvettes. With these parameters, three plasmids (pSU2718, pCML, pEV3) with molecular sizes of 2.6, 5 and 13.7 kb, respectively were successfully transformed into both pathogens. V. anguillarum isolates belonging to serotypes O1 and O2 were transformed with greatest efficiency, 2.5 × 10³ transformants per μg DNA, being achieved in the serotype O2 strains using plasmid pCML. Strains of serotype O3 were not transformed. In the case of P. piscicida the maximum efficiency achieved was 9.8 × 10² transformants per μg pCML plasmid DNA. This optimized system will allow development of procedures for the genetic manipulation of these pathogens.     

Age-associated changes in skeletal muscles and their effect on mobility: an operational diagnosis of sarcopenia.

Sarcopenia, the reduction of muscle mass and strength that occurs with aging, is widely considered one of the major causes of disability in older persons. Surprisingly, criteria that may help a clinician to identify persons with impaired muscle function are still lacking. Using data from a large representative sample of the general population, we examined how muscle function and calf muscle area change with aging and affect mobility in men and women free of neurological conditions. We tested several putative indicators of sarcopenia, including knee extension isometric torque, handgrip, lower extremity muscle power, and calf muscle area. For each indicator, sarcopenia was considered to be present when the measure was >2 SDs below the mean. For all four measures, the prevalence of sarcopenia increased with age, both in men and women. The age-associated gradient in prevalence was maximum for muscle power and minimum for calf-muscle area. However, lower extremity muscle power was no better than knee-extension torque or handgrip in the early identification of poor mobility, defined either as walking speed <0.8 m/s or inability to walk at least 1 km without difficulty and without developing symptoms. Optimal cutoff values that can be used in the clinical practice to identify older persons with poor mobility were developed. The findings of the study lay the basis for a cost-effective, clinical marker of sarcopenia based on a measure of isometric handgrip strength. Our findings should be verified in a longitudinal study.     

The two manganese peroxidases Pr-MnP2 and Pr-MnP3 of Phlebia radiata, a lignin-degrading basidiomycete, are phylogenetically and structurally divergent

Two new, at primary sequence and protein structure levels different, manganese peroxidase encoding genes from the white rot basidiomycete Phlebia radiata are described. Both genes are expressed in liquid cultures of P. radiata containing milled alder wood or glucose as carbon source, and high Mn(2+) concentration. The gene Pr-mnp2 contains 7 introns and codes for a 390 amino-acid polypeptide, whereas Pr-mnp3 presents 11 introns and codes for a 362 amino-acid protein. The 3-D molecular models confirm this diversity; the predicted Pr-MnP2 with a long C-terminal extension has the highest structural similarity with the crystal structure of Phanerochaete chrysosporium MnP1, whereas the shorter Pr-MnP3 protein is structurally more related to lignin peroxidases (P. chrysosporium LiPH8/H2). In Pr-MnP3, however, an alanine replaces the exposed tryptophan present in LiP and versatile peroxidases, and both Pr-MnPs include the conserved Mn(2+)-binding amino-acid ligands. This is the first occasion when two enzymes of similar function and origin fall into phylogenetically distinct subfamilies within the expanding dendrogram of the class II fungal secretory heme peroxidases.     

Oxidative stress and photoinhibition can be separated in the cyanobacterium Synechocystis sp. PCC 6803

Roles of oxidative stress and photoinhibition in high light acclimation were studied using a regulatory mutant of the cyanobacterium Synechocystis sp. PCC 6803. The mutant strain ΔsigCDE contains the stress responsive SigB as the only functional group 2 σ factor. The ?sigCDE strain grew more slowly than the control strain in methyl-viologen-induced oxidative stress. Furthermore, a fluorescence dye detecting H₂O₂, hydroxyl and peroxyl radicals and peroxynitrite, produced a stronger signal in ?sigCDE than in the control strain, and immunological detection of carbonylated residues showed more protein oxidation in ?sigCDE than in the control strain. These results indicate that ?sigCDE suffers from oxidative stress in standard conditions. The oxidative stress may be explained by the findings that ?sigCDE had a low content of glutathione and low amount of Flv3 protein functioning in the Mehler-like reaction. Although ?sigCDE suffers from oxidative stress, up-regulation of photoprotective carotenoids and Flv4, Sll2018, Flv2 proteins protected PSII against light induced damage by quenching singlet oxygen more efficiently in ?sigCDE than in the control strain in visible and in UV-A/B light. However, in UV-C light singlet oxygen is not produced and PSII damage occurred similarly in the ?sigCDE and control strains. According to our results, resistance against the light-induced damage of PSII alone does not lead to high light tolerance of the cells, but in addition efficient protection against oxidative stress would be required.     

Thermotolerant and thermostable laccases

Laccases are phenol-oxidizing, usually four-copper containing metalloenzymes. For industrial and biotechnological purposes, laccases were among the first fungal oxidoreductases providing larger-scale applications such as removal of polyphenols in wine and beverages, conversion of toxic compounds and textile dyes in waste waters, and in bleaching and removal of lignin from wood and non-wood fibres. In order to facilitate novel and more efficient bio-catalytic process applications, there is a need for laccases with improved biochemical properties, such as thermostability and thermotolerance. This review gives a current overview on the sources and characteristics of such laccases, with particular emphasis on the fungal enzymes.     

Kinetics of prolonged photoinhibition revisited: photoinhibited Photosystem II centres do not protect the active ones against loss of oxygen evolution

Photoinhibition of Photosystem II (PSII) in lincomycin-treated leaves begins as a first-order reaction, but fluorescence measurements have suggested that after prolonged illumination, the number of active PSII centres stabilizes to 15–20% of control. The stabilization has been interpreted to indicate that photoinhibited PSII centres protect the remaining active centres against photoinhibition (Lee, Hong and Chow, Planta 212:332–342, 2001). In an attempt to study the mechanism of this protection, we measured the reaction kinetics of photoinhibition in lincomycin-treated pumpkin (Cucurbita pepo L.) and pepper (Capsicum annuum L.) leaves in vivo. The light-saturated rate of PSII oxygen evolution, assayed from thylakoids and isolated from the treated leaves, was used as a direct measure of the number of remaining active PSII centres, and the fluorescence parameters F _V/F _M and (F _V/F _M)/F ₀ (=1/F ₀ − 1/F _M) were measured for comparison. To our surprise, no stabilization of PSII activity was observed and photoinhibition followed first-order kinetics until PSII activity had virtually declined to zero. A series of in vitro experiments was carried out to see whether stabilization of PSII activity occurs if a particular combination of light intensity and wavelength range is applied, or if a specific PSII preparation is used as experimental material. The results of the in vitro experiments confirmed the in vivo result about persistent first-order kinetics. We conclude that photoinhibited PSII centres offer no measurable protection against photoinhibition.