Characterization of the dTDP-Fuc3N and dTDP-Qui3N biosynthetic pathways in Campylobacter jejuni 81116.

The Gram-negative bacterium Campylobacter jejuni 81116 (Penner serotype HS:6) has a class E lipooligosaccharide (LOS) biosynthesis locus containing 19 genes, which encode for 11 putative glycosyltransferases, 1 lipid A acyltransferase and 7 enzymes thought to be involved in the biosynthesis of dideoxyhexosamine (ddHexN) moieties. Although the LOS outer core structure of C. jejuni 81116 is still unknown, recent mass spectrometry analyses suggest that it contains acetylated forms of two ddHexN residues. For this investigation, five of the genes encoding enzymes reportedly involved in the biosyntheses of these sugar residues were examined, rmlA, rmlB, wlaRA, wlaRB and wlaRG. Specifically, these genes were cloned and expressed in Escherichia coli, and the corresponding enzymes were purified and tested for biochemical activity. Here we present data demonstrating that RmlA functions as a glucose-1-phosphate thymidylyltransferase and that RmlB is a thymidine diphosphate (dTDP)-glucose 4,6-dehydratase. We also show, through nuclear magnetic resonance spectroscopy and mass spectrometry analyses, that WlaRG, when utilized in coupled assays with either WlaRA or WlaRB and dTDP-4-keto-6-deoxyglucose, results in the production of either dTDP-3-amino-3,6-dideoxy-d-galactose (dTDP-Fuc3N) or dTDP-3-amino-3,6-dideoxy-d-glucose (dTDP-Qui3N), respectively. In addition, the X-ray crystallographic structures of the 3,4-ketoisomerases, WlaRA and WlaRB, were determined to 2.14 and 2.0 Å resolutions, respectively. Taken together, the data reported herein demonstrate that C. jejuni 81116 utilizes five enzymes to synthesize dTDP-Fuc3N or dTDP-Qui3N and that WlaRG, an aminotransferase, can function on sugars with differing stereochemistry about their C-4' carbons. Importantly, the data reveal that C. jejuni 81116 has the ability to synthesize two isomeric ddHexN forms.

Dietary fish oil substitution alters the eicosanoid profile in ankle joints of mice during Lyme infection.

Dietary ingestion of (n-3) PUFA alters the production of eicosanoids and can suppress chronic inflammatory and autoimmune diseases. The extent of changes in eicosanoid production during an infection of mice fed a diet high in (n-3) PUFA, however, has not, to our knowledge, been reported. We fed mice a diet containing either 18% by weight soybean oil (SO) or a mixture with fish oil (FO), FO:SO (4:1 ratio), for 2 wk and then infected them with Borrelia burgdorferi. We used an MS-based lipidomics approach and quantified changes in eicosanoid production during Lyme arthritis development over 21 d. B. burgdorferi infection induced a robust production of prostanoids, mono-hydroxylated metabolites, and epoxide-containing metabolites, with 103 eicosanoids detected of the 139 monitored. In addition to temporal and compositional changes in the eicosanoid profile, dietary FO substitution increased the accumulation of 15-deoxy PGJ(2), an antiinflammatory metabolite derived from arachidonic acid. Chiral analysis of the mono-hydroxylated metabolites revealed they were generated from primarily nonenzymatic mechanisms. Although dietary FO substitution reduced the production of inflammatory (n-6) fatty acid-derived eicosanoids, no change in the host inflammatory response or development of disease was detected.

Chemoorganotrophic Bacteria From Lake Fryxell, Antarctica, Including Pseudomonas Strain LFY10, a Cold-Adapted, Halotolerant Bacterium Useful in Teaching Labs.

Lake Fryxell, situated in the McMurdo Dry Valleys of Antarctica, is an intriguing aquatic ecosystem because of its perennial ice cover, highly stratified water column, and extreme physicochemical conditions, which collectively restrict lake biodiversity to solely microbial forms. To expand our current understanding of the cultivable biodiversity of Lake Fryxell, water samples were collected from depths of 10 and 17 m, and pure cultures of eight diverse strains of aerobic, chemoorganotrophic bacteria were obtained. Despite having high 16S rRNA gene sequence similarity to mesophilic bacteria inhabiting various temperate environments, all Lake Fryxell isolates were psychrotolerant, with growth occurring at 0°C and optimal growth from 18-24°C for all isolates. Phylogenetic analyses showed the isolates to be members of six taxonomic groups, including the genera Brevundimonas, Arthrobacter, Sphingobium, Leifsonia, and Pseudomonas, as well as the family Microbacteriaceae (one strain could not reliably be assigned to a specific genus based on our analysis). Pseudomonas strain LFY10 stood out as a useful tool for teaching laboratory activities because of its substantial cold adaptation (visible growth is evident in 1-2 days at 4°C), beta-hemolytic activity, and halotolerance to 8.5% (w/v) NaCl. These cold-adapted bacteria likely play a role in carbon mineralization and other nutrient cycling in Lake Fryxell, and their characterization broadens our understanding of microbial biodiversity in aquatic polar ecosystems.

A Laboratory Activity Demonstrating the Antibacterial Effects of Extracts from Two Plant Species, Moringa oleifera and Allium sativum (Garlic).

A variety of plants synthesize natural products that either kill or inhibit the growth of various microorganisms. These plant products may serve as useful natural alternatives to synthetic antimicrobial pharmaceuticals and can be especially important in regions where commercial drugs are often not available. Despite this, the role of plants as producers of natural antimicrobial agents is often understated or even ignored in undergraduate biology curricula. In this laboratory exercise, students extract water-soluble constituents from two plants, Moringa oleifera (moringa) and Allium sativum (garlic), and determine their activity against both a gram-positive (Bacillus cereus strain 971) and a gram-negative (Escherichia coli strain K12) bacterium using a disk diffusion assay on Mueller-Hinton agar. Disks infused with commercially available antibiotics (e.g., penicillin and tetracycline) serve as controls. Following an incubation period of 24 hours, students obtain quantitative data by measuring zones of growth inhibition that develop as a result of strain sensitivity. To determine the effectiveness of the learning objectives, an unannounced quiz was administered both before and after the activity, and the students showed significant gains in their understanding of key concepts. Because this activity combines aspects of two major branches of biology-plant biology and microbiology-it is suitable for use as a laboratory exercise in courses related to either discipline, or it may be used as a laboratory component of a general biology course.

Cold-Active, Heterotrophic Bacteria from the Highly Oligotrophic Waters of Lake Vanda, Antarctica.

The permanently ice-covered lakes of the McMurdo Dry Valleys, Antarctica are distinctive ecosystems that consist strictly of microbial communities. In this study, water samples were collected from Lake Vanda, a stratified Dry Valley lake whose upper waters (from just below the ice cover to nearly 60 m) are highly oligotrophic, and used to establish enrichment cultures. Six strains of psychrotolerant, heterotrophic bacteria were isolated from lake water samples from a depth of 50 or 55 m. Phylogenetic analyses showed the Lake Vanda strains to be species of Nocardiaceae, Caulobacteraceae, Sphingomonadaceae, and Bradyrhizobiaceae. All Lake Vanda strains grew at temperatures near or below 0 °C, but optimal growth occurred from 18 to 24 °C. Some strains showed significant halotolerance, but no strains required NaCl for growth. The isolates described herein include cold-active species not previously reported from Dry Valley lakes, and their physiological and phylogenetic characterization broadens our understanding of these limnologically unique lakes.

The intrinsic cysteine and histidine residues of the anti-Salmonella antibody Se155-4: a model for the introduction of new functions into antibody-binding sites.

New functions can be incorporated into anti-hapten or anti-protein antibodies by mutating selected residues in the binding-site region either to Cys, to allow alkylation with reagents bearing the desired functional groups, or to His, to create metal-binding sites or to make antigen binding pH-sensitive. However, choosing suitable sites for these mutations has been hampered by the lack of antibodies with these features, to serve as models. Remarkably, the anti-carbohydrate antibody Se155-4, specific for the Salmonella group B lipopolysaccharide, already has a Cys and two pairs of His residues close to the antigen-binding pocket in its structure, and shows pH-dependent antigen binding. We therefore investigated modification of its Cys94L in an scFv version of the antibody with the aims of creating a 'reagentless' fluorescent sensor and attaching a metal-binding group that might confer lyase activity. These groups were successfully introduced, as judged by mass spectrometry, and had only slightly reduced antigen binding in enzyme-linked immunosorbent assay. The fluorescent product was sensitive to addition of antigen in a solution format, unlike a modification of a more distant Cys introduced into the VH CDR4 loop. Two other routes to modulate antigen binding were also explored, metal binding by the His pair alongside the antigen-binding pocket and insertions into CDR4 to extend the antigen-contact area. His residues adjacent to the antigen-binding pocket bound copper, causing a 5-fold decrease in antigen binding. In CDR4 of the VH domain, the preferred insert length was four residues, which gave stable antigen-binding products but did not improve overall antigen affinity.