New convenient defined media for [(35)S]methionine labelling and proteomic analyses of probiotic lactobacilli.

AIMS: To develop experimental conditions for efficient protein radiolabelling and two-dimensional (2D) polyacrylamide gel electrophoresis for investigation of stress proteomes of probiotic Lactobacillus spp. METHODS AND RESULTS: Three chemically defined media (CDM) optimized from a commercial medium supported rapid growth of the probiotic Lactobacillus rhamnosus E97800, Lactobacillus brevis ATCC 8287 and Lactobacillus reuteri E97849, and a broad range of other lactic acid bacteria. These CDM allowed efficient protein radiolabelling, requiring as little as 200 mul of logarithmic culture and pulse-chase labelling of 20 min to detect c. 300 distinct protein spots in a mini-scale 2D-gel. Proteins including DnaK, GroEL and ClpATPases were identified from the 2D-gels by immunoblotting. CONCLUSIONS: Radiolabelling coupled with 2D gel electrophoresis provides a sensitive means to monitor changes in protein synthesis rates in probistic lactobacilli. SIGNIFICANCE AND IMPACT OF THE STUDY: Efficient tools for proteomic analyses of probiotic Lactobacillus were developed and applied for stress-response studies.

Diversity of endophytic fungi of single Norway spruce needles and their role as pioneer decomposers.

The diversity of endophytic fungi within single symptomless Norway spruce needles is described and their possible role as pioneer decomposers after needle detachment is investigated. The majority (90%) of all 182 isolates from green intact needles were identified as Lophodermium piceae. Up to 34 isolates were obtained from single needles. Generally, all isolates within single needles had distinct randomly amplified microsatellite (RAMS) patterns. Single trees may thus contain a higher number of L. piceae individuals than the number of their needles. To investigate the ability of needle endophytes to act as pioneer decomposers, surface-sterilized needles were incubated on sterile sand inoculated with autoclaved or live spruce forest humus layer. The dry weight loss of 13-17% found in needles after a 20-week incubation did not significantly differ between the sterilized and live treatments. Hence, fungi surviving the surface sterilization of needles can act as pioneer decomposers. A considerable portion of the needles remained green during the incubation. Brown and black needles, in which the weight loss had presumably taken place, were invaded throughout by single haplotypes different from L. piceae. Instead, Tiarasporella parca, a less common needle endophyte, occurred among these invaders of brown needles. Needle endophytes of Norway spruce seem thus to have different abilities to decompose host tissues after needle cast. L. piceae is obviously not an important pioneer decomposer of Norway spruce needles. The diversity of fungal individuals drops sharply when needles start to decompose. Thus, in single needles the decomposing mycota is considerably less diverse than the endophytic mycota.

Specific binding of baculoviruses displaying gp64 fusion proteins to mammalian cells.

Viral vectors displaying specific ligand binding moieties have raised an increasing interest in the area of targeted gene therapy. In this report, we describe baculovirus vectors displaying either a functional single chain antibody fragment (scFv) specific for the carcinoembryonic antigen (CEA) or the synthetic IgG binding domains (ZZ) derived from protein A of Staphylococcus aureus. In addition, the vectors were engineered to incorporate a reporter gene encoding the enhanced green fluorescent protein (EGFP) under the transcriptional regulation of the cytomegalovirus (CMV) IE promoter. Display of the targeting moieties on the viral surface was achieved through fusion to the N-terminus of gp64, the major envelope protein of the Autographa californica nuclear polyhedrosis virus (AcNPV). Specific binding of the gp64 fusion viruses to mammalian target cells was demonstrated by using monoclonal anti-gp64 antibodies followed by fluorescence and/or confocal microscopy. The anti-CEA scFv displaying baculovirus was shown to bind specifically to CEA expressing cells (PC-3). Similarly, the virus displaying the ZZ domains of protein A was targeted to BHK cells via binding of an appropriate IgG antibody. In all cases, the reporter gene was expressed in the transduced mammalian cells as shown by fluorescence microscopy and flow cytometric analyses.