Identification of Lactobacillus reuteri Genes Specifically Induced in the Mouse Gastrointestinal Tract

Lactobacilli are common inhabitants of the gastrointestinal tracts of mammals and have received considerable attention due to their putative health-promoting properties. Little is known about the traits that enhance the ability of these bacteria to inhabit the gastrointestinal tract. In this paper we describe the development and application of a strategy based on in vivo expression technology (IVET) that enables detection of Lactobacillus reuteri genes specifically induced in the murine gut. A plasmid-based system was constructed containing ′ermGT (which confers lincomycin resistance) as the primary reporter gene for selection of promoters active in the gastrointestinal tract of mice treated with lincomycin. A second reporter gene, ′bglM (β-glucanase), allowed differentiation between constitutive and in vivo inducible promoters. The system was successfully tested in vitro and in vivo by using a constitutive promoter. Application of the IVET system with chromosomal DNA of L. reuteri 100-23 and reconstituted lactobacillus-free mice revealed three genes induced specifically during colonization. Two of the sequences showed homology to genes encoding xylose isomerase (xylA) and peptide methionine sulfoxide reductase (msrB), which are involved in nutrient acquisition and stress responses, respectively. The third locus showed homology to the gene encoding a protein whose function is not known. Our IVET system has the potential to identify genes of lactobacilli that have not previously been functionally characterized but which may be essential for growth of these bacteria in the gastrointestinal ecosystem.     

A comparison of four different fine root production estimates with ecosystem carbon balance data in a Fagus–Quercus mixed forest

The controversy on how to measure fine root production of forests (P) most accurately continues. We applied four different approaches to determine annual rates of P in an old-growth temperate Fagus sylvatica–Quercus petraea stand: sequential soil coring with minimum–maximum calculation, sequential coring with compartmental flow calculation, the ingrowth core method, and a recently developed root chamber method for measuring the growth of individual fine roots in situ. The results of the four destructive approaches differed by an order of magnitude and, thus, are likely to introduce large errors in estimating P. The highest annual rates of P were obtained from the sequential coring approach with compartmental flow calculation, intermediate rates by sequential coring with minimum–maximum calculation, and low ones by both the root growth chamber and ingrowth core approaches. A carbon budget for the stand was set up based on a model of annual net carbon gain by the canopy and measurements on carbon sink strength (annual leaf, branch and stem growth). The budget implied that a maximum of 27% of the net carbon gain was available for allocation to fine root growth. When compared to the carbon budget data, the sequential coring/compartmental flow approach overestimated annual fine root production substantially; whereas the ingrowth core and root growth chamber approaches grossly underestimated P rates. With an overestimation of about 25% the sequential coring/minimum–maximum approach demonstrated the best agreement with the carbon budget data. It is concluded that the most reliable estimate of P in this temperate forest will be obtained by applying the sequential coring/minimum–maximum approach, conducted with a large number of replicate samples taken on a few dates per season, in conjunction with direct root growth observation by minirhizotrons.     

Purification and characterization of chorismate mutase isoenzymes from ruta graveolens l.

Two isoenzymes of chorismate mutase (EC 5.4.99.5), designated as CM-1 and CM-2, were isolated and partially purified from suspension-cultured cells of Ruta gravelens by DEAE-sephacel chromatography and gel filtration. 60–72% of the total activity measured after DEAE-sephacel chromatography were obtained as CM-1 and 28–40% were CM-2 activity. CM-1 was inhibited by phenylalanine (K₁ = 4 · 10^?6 M) and tyrosine (K₁ = 8. 10^?6M) and activated by tryptophan. In contrast, CM-2 was not influenced by these three amino acids. The molecular weights estimated by gel filtration on SEPHADEX G-150 were 56000 for CM-1 and 45000 for CM-2, respectively. Both isoenzymes were stable at ?20°C, but exhibited different behaviour during thermal inactivation and different optima of reaction temperature. CM-1 catalysed the reaction at a pH optimum of pH 7.8 and CM-2 showed a broad optimum between 6–10. The K_m-values for chorismic acid were determined to be 1.1 mM for CM-1 and 0.5 mM for CM-2. The isoenzymes showed different behaviour to inhibitors of sulfhydryl groups. There were no differences in all parameters of chorismate mutase examined for two various cell lines of Ruta graveolens.     

The High-Mobility Group Protein T160 Binds to both Linear and Cruciform DNA and Mediates DNA Bending as Determined by Ring Closure

The high-mobility group protein T160 was isolated by screening a phage library from a murine pre-B-cell line L1210. South–Western experiments have previously shown that this protein binds to V-(D)-J recombination signal sequences, suggesting that it may be a sequence-specific DNA-binding protein. However, neither gel-shift nor footprinting analyses have been successfully employed with the T160 protein, despite an extensive effort. In this study, the T160 protein or truncated forms made soluble through denaturing and renaturing cycles in urea were successfully used in gel-shift experiments showing that T160 binds to cruciform or linear duplex DNA with no apparent sequence specificity. Furthermore, fragments longer than 100 bp efficiently formed covalently closed circular monomers in the presence of T160 and T4 DNA ligase, indicating that the protein is capable of introducing bends into the duplex. Last, tissue distribution by Western blotting analysis showed that the T160 protein is expressed in various murine tissues in addition to those of lymphoid origin. Considering its broad evolutionary conservation (from plants to mammals) also, these results suggest that the functional role of the T160 protein is not limited to V-(D)-J recombination, but might be involved in basic processes such as DNA replication and repairing, where irregular DNA structures are generated and very likely recognized by HMG domain proteins.