Identification of a novel glycosyltransferase involved in LOS biosynthesis of Moraxella catarrhalis

Moraxella catarrhalis is an important human mucosal pathogen that contributes to otitis media in infants and exacerbates conditions such as chronic obstructive pulmonary disease in the elderly. This study describes the identification of a novel gene, lgt5 that encodes a glycosyltransferase involved in the LOS biosynthesis of M. catarrhalis. Analysis of NMR data of LOS-derived oligosaccharide from a Serotype A lgt5 mutant strain of M. catarrhalis indicate that lgt5 encodes an alpha-(1-->4)-galactosyltransferase.     

Evaluation of Escherichia coli K-12 343/113 and derived strains for microbial mutagenicity assays

Several characteristics of the E. coli K-12 mutagenicity tester strains 343/113 and 343/120 have been investigated for their effects on induced mutagenesis using the arg56 and nad113 genes, and resistance to valine. We found, as have earlier authors, that the nad113 marker is relatively specific for detecting frameshift-inducing mutagens and relatively insensitive to agents that cause point mutations. In contrast, both the Arg and Val markers are primarily specific for reversion (or mutation) induction by point mutagens. In all cases tested, the Arg and Val markers respond to mutagens in a qualitatively similar manner. We have enhanced the sensitivity of this tester system to a wide variety of mutagens by permeabilization of the tester cell population using Tris-EDTA treatment. This treatment prior to mutagen exposure enables the detection of mutagenicity of several compounds that are weakly mutagenic or nonmutagenic in untreated cells. We have also increased the mutagenicity of some chemicals by preincubating with rat-liver S9 at pH values other than 7.4. For diethylnitrosamine, for instance, maximal induction occurred at pH 6.5, and for benzo[a]pyrene, maximal induction was at pH 6.8.     

Immobilized pH gradient-driven paper-based IEF: a new method for fractionating complex peptide mixtures before MS analysis

Introduction  

The vast difference in the abundance of different proteins in biological samples limits the determination of the complete proteome of a cell type, requiring fractionation of proteins and peptides before MS analysis.     

Comparative Studies on Plastoquinones: V. Changes in Lipophilic Chloroplast Quinones during Development

Changes of lipophilic chloroplast quinones in corn, oats, peas, and Vicia faba are reported after 0, 4, 8, 12, 16, 20, 24, 48, 72, or 96 hours of exposure to light. There is a pronounced increase in plastoquinone A and chlorophyll levels and slight increase, in plastoquinone C_1-6, vitamin K₁, and α-tocopherylquinone content. Coenzyme Q levels, on the other hand, show little change upon exposure to light.     

Characterization of Aerosolized Bacteria and Fungi From Desert Dust Events in Mali, West Africa

Millions of metric tons of African desert dust blow across the Atlantic Ocean each year, blanketing the Caribbean and southeastern United States. Previous work in the Caribbean has shown that atmospheric samples collected during dust events contain living microbes, including plant and opportunistic human pathogens. To better understand the potential downwind public health and ecosystem effects of the dust microbes, it is important to characterize the source population. We describe 19 genera of bacteria and 3 genera of fungi isolated from air samples collected in Mali, a known source region for dust storms, and over which large dust storms travel.     

Catalysis by Glomerella cingulata cutinase requires conformational cycling between the active and inactive states of its catalytic triad

Cutinase belongs to a group of enzymes that catalyze the hydrolysis of esters and triglycerides. Structural studies on the enzyme from Fusarium solani have revealed the presence of a classic catalytic triad that has been implicated in the enzyme's mechanism. We have solved the crystal structure of Glomerella cingulata cutinase in the absence and in the presence of the inhibitors E600 (diethyl p-nitrophenyl phosphate) and PETFP (3-phenethylthio-1,1,1-trifluoropropan-2-one) to resolutions between 2.6 and 1.9 Å. Analysis of these structures reveals that the catalytic triad (Ser136, Asp191, and His204) adopts an unusual configuration with the putative essential histidine His204 swung out of the active site into a position where it is unable to participate in catalysis, with the imidazole ring 11 Å away from its expected position. Solution-state NMR experiments are consistent with the disrupted configuration of the triad observed crystallographically. H204N, a site-directed mutant, was shown to be catalytically inactive, confirming the importance of this residue in the enzyme mechanism. These findings suggest that, during its catalytic cycle, cutinase undergoes a significant conformational rearrangement converting the loop bearing the histidine from an inactive conformation, in which the histidine of the triad is solvent exposed, to an active conformation, in which the triad assumes a classic configuration.     

Gibberellin-like substances and indole type auxins in developing grains of normal- and high-lysine genotypes of barley

Changes in gibberellin-like activity and content of indole type auxins were investigated during grain development of the two high-lysine barley (Hordeum vulgare L.) genotypes Sv 73608 and Risø 1508, and their corresponding normal cultivars Mona and Bomi. A peak in gibberellin-like activity was found in developing grains of the normal cultivars about 18 days after anthesis, whereas the grains of the high-lysine genotypes showed a two to five times higher maximum about 3–4 days later. The auxin content of the cultivar Bomi showed a maximum between the 22nd and the 29th day after anthesis, whereas, throughout their development the grains of the mutant Risø 1508 exhibited only about ¹/10 of the maximum level of auxin found in the grains of Bomi. The normal cultivar Mona also displayed higher contents of auxin than the high-lysine genotypes Sv 73608, particularly at the later stages of grain growth, but the differences in concentration were considerable smaller than for the pair ‘Bomi’—‘Risø 1508’. It is suggested that auxins play an important role in the development of barley grains.     

Functional implications of the expression of PilC proteins in meningococci

Multiple forms of PilC were found in Neisseria meningitidis (Nm) strains isolated from the oropharynx, blood or cerebrospinal fluid expressing either Class I or Class II pili. PilC expression was observed less frequently in case as opposed to carrier isolates. Moreover, PilC and pili were not always co-expressed. Several heavily piliated strains had no detectable PilC protein as determined by Western blotting using an antiserum previously used to detect such proteins in adhesive variants (Nassif et al., 1994). Serogroup B strain MC58 produced large numbers of pili, but expressed barely detectable amounts of PilC. A clonal variant of this strain with increased expression of PilC concurrently exhibited increased adherence to Chang conjunctival epithelial cells and human umbilical vein endothelial cells (Huvecs), but with more rapid binding to the former. No alteration in pilin sequence occurred in this variant, suggesting the involvement of PilC in increased adhesion. A Pil^- backswitcher isolated from the hyper-adherent variant was PilC⁺ but was non-adherent, indicating that any PilC adherence function requires pilus expression. Parental variant (low PilC) produced pili in bundles that were easily detached from the bacterial surface and were frequently associated with Huvec surfaces after bacteria had been sheared off, but pili infrequently replaced bacteria during infection with the PilC-expressing variant. The hyper-adherent variant, which appeared to produce morphologically distinct pilus bundles, was able to withstand considerable shearing force and remained firmly attached to Huvecs. This raises the possibility that the observed hyper-adherence may arise from better anchorage of pili to the bacterial surface in addition to increased adhesion to some host cell surfaces.