To unravel the biological function of the widely used probiotic bacterium Lactobacillus rhamnosus GG, we compared its 3.0-Mbp genome sequence with the similarly sized genome of L. rhamnosus LC705, an adjunct starter culture exhibiting reduced binding to mucus. Both genomes demonstrated high sequence identity and synteny. However, for both strains, genomic islands, 5 in GG and 4 in LC705, punctuated the colinearity. A significant number of strain-specific genes were predicted in these islands (80 in GG and 72 in LC705). The GG-specific islands included genes coding for bacteriophage components, sugar metabolism and transport, and exopolysaccharide biosynthesis. One island only found in L. rhamnosus GG contained genes for 3 secreted LPXTG-like pilins (spaCBA) and a pilin-dedicated sortase. Using anti-SpaC antibodies, the physical presence of cell wall-bound pili was confirmed by immunoblotting. Immunogold electron microscopy showed that the SpaC pilin is located at the pilus tip but also sporadically throughout the structure. Moreover, the adherence of strain GG to human intestinal mucus was blocked by SpaC antiserum and abolished in a mutant carrying an inactivated spaC gene. Similarly, binding to mucus was demonstrated for the purified SpaC protein. We conclude that the presence of SpaC is essential for the mucus interaction of L. rhamnosus GG and likely explains its ability to persist in the human intestinal tract longer than LC705 during an intervention trial. The presence of mucus-binding pili on the surface of a nonpathogenic Gram-positive bacterial strain reveals a previously undescribed mechanism for the interaction of selected probiotic lactobacilli with host tissues.genome ͉ probiotics ͉ adhesion ͉ pilus ͉ lactic acid bacteria

The filamentous fungus Trichoderma reesei has tremendous capability to secrete proteins. Therefore, it would be an excellent host for producing high levels of therapeutic proteins at low cost. Developing a filamentous fungus to produce sensitive therapeutic proteins requires that protease secretion is drastically reduced. We have identified 13 major secreted proteases that are related to degradation of therapeutic antibodies, interferon alpha 2b, and insulin like growth factor. The major proteases observed were aspartic, glutamic, subtilisin-like, and trypsin-like proteases. The seven most problematic proteases were sequentially removed from a strain to develop it for producing therapeutic proteins. After this the protease activity in the supernatant was dramatically reduced down to 4% of the original level based upon a casein substrate. When antibody was incubated in the six protease deletion strain supernatant, the heavy chain remained fully intact and no degradation products were observed. Interferon alpha 2b and insulin like growth factor were less stable in the same supernatant, but full length proteins remained when incubated overnight, in contrast to the original strain. As additional benefits, the multiple protease deletions have led to faster strain growth and higher levels of total protein in the culture supernatant.

The cell surface is covered by a dense layer of protein-and lipid-linked glycans. Although it has been known that distinct glycan structures are associated with cancer, the whole spectrum of cancer-associated glycans has remained undiscovered. In the present study, we analyzed the protein-linked cancer glycome by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric glycan profiling of cancer patient tissue samples. In lung cancer, we detected accumulation of a novel group of tumor-associated glycans. These protein-linked glycans carried abnormal nonreducing terminal B-N-acetyl-D-glucosamine (GlcNAc) residues. A similar phenomenon was also detected in structural analyses of tumor-derived glycosphingolipids. This showed that glycan biosynthesis may dramatically change in cancer and that direct glycome analysis can detect the resulting marker glycans. Based on the structural knowledge, we further devised a covalent labeling technique for the detection of GlcNAcexpressing tumors with a specific transferase enzyme. In normal tissues, terminal GlcNAc antigens are capped by galactosylation. Similarly to common cancer-associated glycan antigens T, Tn, and sialyl-Tn, the newly discovered GlcNAc antigens result from incomplete glycosylation. In conclusion, the identified terminal GlcNAc glycans should be recognized as a novel class of tumor markers. [Cancer Res 2009;69(14):5811-9]

Lactose is the only soluble and economically feasible carbon source for the production of cellulases or heterologous proteins regulated by cellulase expression signals by Hypocrea jecorina (Trichoderma reesei). We investigated the role of the major ␤-galactosidase of H. jecorina in lactose metabolism and cellulase induction. A genomic copy of the bga1 gene was cloned, and this copy encodes a 1,023-amino-acid protein with a 20-amino-acid signal sequence. This protein has a molecular mass of 109.3 kDa, belongs to glycosyl hydrolase family 35, and is the major extracellular ␤-galactosidase during growth on lactose. Its transcript was abundant during growth on L-arabinose and L-arabinitol but was much less common when the organism was grown on lactose, D-galactose, galactitol, D-xylose, and xylitol. ⌬bga1 strains grow more slowly and accumulate less biomass on lactose, but the cellobiohydrolase I and II gene expression and the final cellulase yields were comparable to those of the parental strain. Overexpression of bga1 under the control of the pyruvate kinase promoter reduced the lag phase, increased growth on lactose, and limited transcription of cellobiohydrolases. We detected an additional extracellular ␤-galactosidase activity that was not encoded by bga1 but no intracellular ␤-galactosidase activity. In conclusion, cellulase production on lactose occurs when ␤-galactosidase activity levels are low but decreases as the ␤-galactosidase activities increase. The data indicate that bga1-encoded ␤-galactosidase activity is a critical factor for cellulase production on lactose.The ascomycete Hypocrea jecorina (anamorph, Trichoderma reesei) is used industrially to produce cellulolytic and hemicellulolytic enzymes, and its strong cellulase promoters are of interest for heterologous protein production by this fungus. Cellulase and hemicellulase formation can be induced by several mono-and disaccharides, including sophorose, xylobiose, lactose, D-xylose, and L-sorbose, most of which are too expensive for industrial fermentations. Thus, the range of technically applicable carbon sources is limited. For cellulase and heterologous protein production with cellulase promoters, lactose (1,4-O-␤-D-galactopyranosyl-D-glucose) is virtually the only carbon source that can be used. However, the mechanism by which lactose triggers cellulase formation is not understood. Lactose metabolism is slow, and the cellulase yields on lactose are lower than those on cellulose (27, 28).A key enzyme in the induction of cellulase is galactokinase, since a galactokinase-negative mutant cannot induce cellulases during growth on lactose (30). Lactose can enter the cell after it is hydrolyzed extracellularly to D-glucose and D-galactose by a ␤-galactosidase (lactase, ␤-D-galactoside galactohydrolase; E.C 3.2.1.23), and the monosaccharides are then taken up by the respective permeases. Extracellular ␤-galactosidases are known from several fungi, including Aspergillus niger, Aspergillus oryzae, and Penicillium canescens (5,21,26,29). Alternatively, the...

Leuconostoc gasicomitatum is a psychrotrophic lactic acid bacterium causing spoilage of cold-stored, modified-atmosphere-packaged (MAP), nutrient-rich foods. Its role has been verified by challenge tests in gas and slime formation, development of pungent acidic and buttery off odors, and greening of beef. MAP meats have especially been prone to L. gasicomitatum spoilage. In addition, spoilage of vacuum-packaged vegetable sausages and marinated herring has been reported. The genomic sequencing project of L. gasicomitatum LMG 18811T was prompted by a need to understand the growth and spoilage potentials of L. gasicomitatum, to study its phylogeny, and to be able to knock out and overexpress the genes. Comparative genomic analysis was done within L. gasicomitatum LMG 18811T and the three fully assembled Leuconostoc genomes (those of Leuconostoc mesenteroides, Leuconostoc citreum, and Leuconostoc kimchii) available. The genome of L. gasicomitatum LMG 18811 T is plasmid-free and contains a 1,954,080-bp circular chromosome with an average GC content of 36.7%. It includes genes for the phosphoketolase pathway and alternative pathways for pyruvate utilization. As interesting features associated with the growth and spoilage potential, LMG 18811 T possesses utilization strategies for ribose, external nucleotides, nucleosides, and nucleobases and it has a functional electron transport chain requiring only externally supplied heme for respiration. In respect of the documented specific spoilage reactions, the pathways/genes associated with a buttery off odor, meat greening, and slime formation were recognized. Unexpectedly, genes associated with platelet binding and collagen adhesion were detected, but their functionality and role in food spoilage and processing environment contamination need further study.