Relationship between Glycolysis and Exopolysaccharide Biosynthesis in Lactococcus lactis

The relationships between glucose metabolism and exopolysaccharide (EPS) production in a Lactococcus lactis strain containing the EPS gene cluster (Eps⁺) and in nonproducer strain MG5267 (Eps⁻) were characterized. The concentrations of relevant phosphorylated intermediates in EPS and cell wall biosynthetic pathways or glycolysis were determined by ³¹P nuclear magnetic resonance. The concentrations of two EPS precursors, UDP-glucose and UDP-galactose, were significantly lower in the Eps⁺ strain than in the Eps⁻ strain. The precursors of the peptidoglycan pathway, UDP-N-acetylglucosamine and UDP-N-acetylmuramoyl-pentapeptide, were the major UDP-sugar derivatives detected in the two strains examined, but the concentration of the latter was greater in the Eps⁺ strain, indicating that there is competition between EPS synthesis and cell growth. An intermediate in biosynthesis of histidine and nucleotides, 5-phosphorylribose 1-pyrophosphate, accumulated at concentrations in the millimolar range, showing that the pentose phosphate pathway was operating. Fructose 1,6-bisphosphate and glucose 6-phosphate were the prominent glycolytic intermediates during exponential growth of both strains, whereas in the stationary phase the main metabolites were 3-phosphoglyceric acid, 2-phosphoglyceric acid, and phosphoenolpyruvate. The activities of relevant enzymes, such as phosphoglucose isomerase, α-phosphoglucomutase, and UDP-glucose pyrophosphorylase, were identical in the two strains. ¹³C enrichment on the sugar moieties of pure EPS showed that glucose 6-phosphate is the key metabolite at the branch point between glycolysis and EPS biosynthesis and ruled out involvement of the triose phosphate pool. This study provided clues for ways to enhance EPS production by genetic manipulation.     

Variability in stream macroinvertebrates at multiple spatial scales

1. We intensively sampled 16 western Oregon streams to characterize: (1) the variability in macroinvertebrate assemblages at seven spatial scales; and (2) the change in taxon richness with increasing sampling effort. An analysis of variance (ANOVA) model calculated spatial variance components for taxon richness, total density, percent individuals of Ephemeroptera, Plecoptera and Trichoptera (EPT), percent dominance and Shannon diversity.
2. At the landscape level, ecoregion and among‐streams components dominated variance for most metrics, accounting for 43–72% of total variance. However, ecoregion accounted for very little variance in total density and 36% of the variance was attributable to differences between streams. For other metrics, variance components were more evenly divided between stream and ecoregion effects.
3. Within streams, approximately 70% of variance was associated with unstructured local spatial variation and not associated with habitat type or transect position. The remaining variance was typically split about evenly between habitat and transect. Sample position within a transect (left, centre or right) accounted for virtually none of the variance for any metric.
4. New taxa per stream increased rapidly with sampling effort with the first four to eight Surber samples (500–1000 individuals counted), then increased more gradually. After counting more than 50 samples, new taxa continued to be added in stream reaches that were 80 times as long as their mean wetted width. Thus taxon richness was highly dependent on sampling effort, and comparisons between sites or streams must be normalized for sampling effort.
5. Characterization of spatial variance structure is fundamental to designing sampling programmes where spatial comparisons range from local to regional scales. Differences in metric responses across spatial scales demonstrate the importance of designing sampling strategies and analyses capable of discerning differences at the scale of interest.     

Bacterial epoxide hydrolase-catalyzed resolution of a 2,2-disubstituted oxirane: optimization and upscaling

The epoxide hydrolase-catalyzed resolution of (±)-2-methylglycidyl benzyl ether, a versatile chiral building block for the asymmetric synthesis of bioactive compounds, mediated by whole cells of Rhodococcus ruber SM 1789 was accomplished. Among various parameters (such as temperature, buffer type, pH and catalyst/substrate-ratio) an elevated substrate-concentration proved to be particularly sensitive with respect to a significant enhancement of the enantioselectivity.     

Synergistic inhibitory activity of α- and β-LFA-1 peptides on LFA-1/ICAM-1 interaction

Interactions of cell-adhesion molecule LFA-1 and its ligand ICAM-1 play important roles during immune and inflammatory responses. Critical residues of LFA-1 for ICAM-1 binding are known to be in the I-domain of the α-subunit and the I-like domain of the β-subunit. On the basis of our previous work demonstrating the inhibitory activity of I-domain cyclic peptide cLAB.L on LFA-1/ICAM-1 interaction, here we have explored the activity of I-like-domain peptide LBE on the binding mechanism of cLAB.L. LBE enhances cLAB.L binding to T-cells and epithelial cells. The adherence of T-cells to epithelial monolayers was suppressed by the two peptides. The addition of LBE to the monolayers prior to the addition cLAB.L produced a better inhibitory effect than the reverse procedure. LBE, but not cLAB.L, changes the ICAM-1 conformation, suggesting that LBE binds to ICAM-1 at sites that are distinct from these of cLAB.L and induces improved conformation in ICAM-1 for binding to cLAB.L.     

Cloning, Sequencing, and Characterization of the Hexahydro-1,3,5-Trinitro-1,3,5-Triazine Degradation Gene Cluster from Rhodococcus rhodochrous

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a high explosive which presents an environmental hazard as a major land and groundwater contaminant. Rhodococcus rhodochrous strain 11Y was isolated from explosive contaminated land and is capable of degrading RDX when provided as the sole source of nitrogen for growth. Products of RDX degradation in resting-cell incubations were analyzed and found to include nitrite, formaldehyde, and formate. No ammonium was excreted into the medium, and no dead-end metabolites were observed. The gene responsible for the degradation of RDX in strain 11Y is a constitutively expressed cytochrome P450-like gene, xplA, which is found in a gene cluster with an adrenodoxin reductase homologue, xplB. The cytochrome P450 also has a flavodoxin domain at the N terminus. This study is the first to present a gene which has been identified as being responsible for RDX biodegradation. The mechanism of action of XplA on RDX is thought to involve initial denitration followed by spontaneous ring cleavage and mineralization.     

The upper cytokine-binding module and the Ig-like domain of the leukaemia inhibitory factor (LIF) receptor are sufficient for a functional LIF receptor complex.

To elucidate the function of the two cytokine-binding modules (CBM) of the leukemia inhibitory factor receptor (LIFR), receptor chimeras of LIFR and the interleukin-6 receptor (IL-6R) were constructed. Either the NH(2)-terminal (chimera RILLIFdeltaI) or the COOH-terminal LIFR CBM (chimera RILLIFdeltaII) were replaced by the structurally related CBM of the IL-6R which does not bind LIF. Chimera RILLIFdeltaI is functionally inactive, whereas RILLIFdeltaII binds LIF and mediates signalling as efficiently as the wild-type LIFR. Deletion mutants of the LIFR revealed that both the NH(2)-terminal CBM and the Ig-like domain of the LIFR are involved in LIF binding, presumably via the LIF site III epitope. The main function of the COOH-terminal CBM of the LIFR is to position the NH(2)-terminal CBM and the Ig-like domain, so that these can bind to LIF. In analogy to a recently published model of the IL-6R complex, a model of the active LIFR complex is suggested which positions the COOH-terminal CBM at LIF site I and the NH(2)-terminal CBM and the Ig-like domain at site III. An additional contact is postulated between the Ig-like domain of gp130 and the NH(2)-terminal CBM of the LIFR.     

Soybean genomic survey: BAC-end sequences near RFLP and SSR markers.

We are building a framework physical infrastructure across the soybean genome by using SSR (simple sequence repeat) and RFLP (restriction fragment length polymorphism) markers to identify BACs (bacterial artificial chromosomes) from two soybean BAC libraries. The libraries were prepared from two genotypes, each digested with a different restriction enzyme. The BACs identified by each marker were grouped into contigs. We have obtained BAC- end sequence from BACs within each contig. The sequences were analyzed by the University of Minnesota Center for Computational Genomics and Bioinformatics using BLAST algorithms to search nucleotide and protein databases. The SSR-identified BACs had a higher percentage of significant BLAST hits than did the RFLP-identified BACs. This difference was due to a higher percentage of hits to repetitive-type sequences for the SSR-identified BACs that was offset in part, however, by a somewhat larger proportion of RFLP-identified significant hits with similarity to experimentally defined genes and soybean ESTs (expressed sequence tags). These genes represented a wide range of metabolic functions. In these analyses, only repetitive sequences from SSR-identified contigs appeared to be clustered. The BAC-end sequences also allowed us to identify microsynteny between soybean and the model plants Arabidopsis thaliana and Medicago truncatula. This map-based approach to genome sampling provides a means of assaying soybean genome structure and organization.