Novel Features of the Polysaccharide-Digesting Gliding Bacterium Flavobacterium johnsoniae as Revealed by Genome Sequence Analysis

The 6.10-Mb genome sequence of the aerobic chitin-digesting gliding bacterium Flavobacterium johnsoniae (phylum Bacteroidetes) is presented. F. johnsoniae is a model organism for studies of bacteroidete gliding motility, gene regulation, and biochemistry. The mechanism of F. johnsoniae gliding is novel, and genome analysis confirms that it does not involve well-studied motility organelles, such as flagella or type IV pili. The motility machinery is composed of Gld proteins in the cell envelope that are thought to comprise the “motor” and SprB, which is thought to function as a cell surface adhesin that is propelled by the motor. Analysis of the genome identified genes related to sprB that may encode alternative adhesins used for movement over different surfaces. Comparative genome analysis revealed that some of the gld and spr genes are found in nongliding bacteroidetes and may encode components of a novel protein secretion system. F. johnsoniae digests proteins, and 125 predicted peptidases were identified. F. johnsoniae also digests numerous polysaccharides, and 138 glycoside hydrolases, 9 polysaccharide lyases, and 17 carbohydrate esterases were predicted. The unexpected ability of F. johnsoniae to digest hemicelluloses, such as xylans, mannans, and xyloglucans, was predicted based on the genome analysis and confirmed experimentally. Numerous predicted cell surface proteins related to Bacteroides thetaiotaomicron SusC and SusD, which are likely involved in binding of oligosaccharides and transport across the outer membrane, were also identified. Genes required for synthesis of the novel outer membrane flexirubin pigments were identified by a combination of genome analysis and genetic experiments. Genes predicted to encode components of a multienzyme nonribosomal peptide synthetase were identified, as were novel aspects of gene regulation. The availability of techniques for genetic manipulation allows rapid exploration of the features identified for the polysaccharide-digesting gliding bacteroidete F. johnsoniae.Flavobacterium johnsoniae (formerly Cytophaga johnsonae) is a member of the large and diverse phylum of gram-negative bacteria known as the Bacteroidetes. Members of this group of organisms have a number of unique characteristics that distinguish them from other bacteria. Some have novel cell surface machinery to utilize polysaccharides (85, 95, 96). Rapid gliding motility over surfaces is also common among these bacteria (59), as are unusual outer membrane sulfonolipids (29) and flexirubin pigments (78). Bacteroidete gene expression and regulation also have novel aspects (10, 11, 20, 39, 92). The many unusual features of these common but understudied bacteria provide numerous avenues for further exploration, which can be greatly aided by analysis of genome sequences.F. johnsoniae digests many polysaccharides and proteins, but it is best known for its ability to rapidly digest insoluble chitin (87). Chitin is one of the most abundant biopolymers on earth (63). F. johnsoniae and other members of the Bacteroidetes phylum are thought to play important roles in the turnover of this compound in many environments (47). F. johnsoniae has become a model system for the study of bacteroidete gliding motility biochemistry and molecular biology (20, 27-29, 59, 72). This paper highlights novel features of the F. johnsoniae genome, with particular emphasis on genes and proteins likely to be involved in polysaccharide utilization, gliding motility, and the novel biochemistry of this organism.     

Metabolic effect of a LoBAG30 diet in men with type 2 diabetes

We recently reported that in subjects with untreated type 2 diabetes a 5-wk diet of 30:30:40 carbohydrate/protein/fat ratio resulted in a significant decrease in 24-h integrated glucose, total %glycohemoglobin, and total cholesterol compared with a control diet of 55:15:30 carbohydrate/protein/fat given at the beginning of the 5-wk period. Body weight was stable and insulin was unchanged. We now present data on other hormones and metabolites considered to be affected by dietary macronutrient changes. The test diet resulted in an elevated fasting plasma total IGF-I, but not growth hormone. Urinary free cortisol was increased. Serum renin and urinary aldosterone remained unchanged. Blood pressure was stable. Serum creatinine and uric acid were increased. Urinary microalbumin was decreased. Creatinine clearance, serum B(12), folate, homocysteine, TSH, and free thyroxine were unchanged. Total triiodothyronine was decreased. Plasma alpha-amino nitrogen, urea nitrogen, and serum albumin were increased. Urea production rate was increased such that a new steady state was present. The calculated urea production rate accounted for 84% of protein ingested on the control diet but only 68% on the test diet, suggesting net nitrogen retention on the latter. Overall, the lack of negative effects, the improved glucose control, and the positive nitrogen balance suggest such a diet will be beneficial for older subjects with type 2 diabetes. Nevertheless, the long-term effects and general applicability of the diet remain to be determined.     

Partitioning of 14C-photoassimllates within seeds of Phaseolus coccineus (Lam.) and Phaseolus coccineus x Phaseolus vulgaris L.

The objective of this study was to determine partitioning within seeds of ¹⁴C-photoassimilates at three stages of seed development in two Phaseolus crosses — P. coccineus Lam. selfed, and P. coccineus x P. vulgaris L. Abortion of the interspecific embryos occurred when the seed reached 10 mm seed length. When expressed as sink strength (% dpm) or sink activity (% dmp/d.wt.) there were no differences in partitioning of ¹⁴C-photoassimilates when whole seeds were analyzed. If the seed was divided into seed coat, liquid endosperm, and embryo, the sink activity of the interspecific embryo was higher than that of the embryo in the selfed seed. Therefore, abortion of these interspecific Phaselus embryos appeared not to be caused by a lack of photoassimilates.Assistant Professor, Professors, respectively.Contribution from the Agr. Expt. Station, University of Minnesota, St. Paul, MN 55108. Paper No. 13,548, Scientific Journal Series. This research was supported in part by the Science and Education Administration of the United States Department of Agriculture under Grant 59-2271-9-2-020-0 and in part by a grant from the Minnesota Soybean Research and Promotion Council.     

Effects of heat shock on the expression of thrombospondin by endothelial cells in culture

Heat-shock proteins from confluent primary cultures of bovine aortic endothelial cells were analyzed by SDS-polyacrylamide gels. In addition to the increased synthesis of the classical heat-shock proteins, there is an increase of a 180,000-mol wt polypeptide in the growth media of heat-shocked cells. Immunoprecipitation with specific antiserum indicates that the 180,000-mol wt polypeptide is thrombospondin. Assay of mRNA levels coding for thrombospondin after brief hyperthermic treatment (45 degrees C, 10 min), followed by a recovery of 2 h at 37 degrees C, results in a twofold increase in mRNA abundance. In contrast, the activation level of the 71,000-mol wt heat-shock protein mRNA occurs at an earlier time than for thrombospondin mRNA. Immunofluorescence microscopy was used to study the intracellular and extracellular distribution of thrombospondin. Thrombospondin is localized to a prominent pattern of granules of intracellular fluorescence in a perinuclear distribution in cells not exposed to heat. Upon heat treatment, the pattern of granules of intracellular fluorescence appears more pronounced, and the fluorescence appears to be clustered more about the nucleus. There are at least three pools of extracellular forms of thrombospondin: (a) the fine fibrillar extracellular matrix thrombospondin; (b) the punctate granular thrombospondin; and (c) the thrombospondin found in the conditioned medium not associated with the extracellular matrix. When bovine aortic endothelial cells are exposed to heat, the extracellular matrix staining of a fibrillar nature is noticeably decreased, with an increase in the number and degree of fluorescence of focal areas where the punctate granule thrombospondin structures are highly localized. No gross morphological changes in extracellular matrix staining of fibronectin was noted. However, the intermediate filament network was very sensitive and collapsed around the nucleus after heat shock. We conclude that the expression of thrombospondin is heat-shock stimulated.     

Restriction fragment length polymorphism of the human T cell receptor alpha gene

Previous studies have demonstrated restriction fragment length polymorphisms (RFLP) in the vicinity of the alpha and beta genes of the human T-cell receptor. In the course of experiments designed to discover additional polymorphic restriction sites, we found a new RFLP of the T-cell alpha gene recognized by the restriction enzyme Taq I. The site was localized to the interval between the most 3 joining (J) exon and the most 5 constant (C) region exon, about 7 kb distant from the previously described Bgl II polymorphic site which mapped to the vicinity of the 3 untranslated exon. With the use of these two polymorphic markers, four Ti-alpha alleles could be identified, allowing unambiguous assignment of all Ti-alpha genes in some families. These markers may be useful in identifying possible immune response genes or disease predisposition genes associated with the genes of the T-cell receptor for antigen.Abbreviations used in this paper RFLP restriction fragment length polymorphism - Ti-alpha alpha gene of the T-cell receptor for antigen     

Mesenchymal-epithelial interactions as factors influencing male accessory sex organ growth in the rat

Mesenchyme (UGM) and epithelium (UGE) isolated from the urogenital sinuses (UGS) of 17-day male and female rat embryos were separated by using a trypsinization procedure, grown on soft agar, transplanted into syngeneic pubertal male hosts as subcapsular renal grafts, and then collected after 29-30 days. Neither UGM nor UGE underwent prostatic morphogenesis when grown under these conditions. However, tissue recombinants composed of UGM + UGE grew and produced prostatic glands with acinar secretory material. Further, UGM + UGE recombinants were made by varying the proportions of mesenchymal and epithelial tissues. The size of the implants was a function of the absolute amount of mesenchyme; increasing the absolute amount of UGM produced larger specimens whereas varying the UGE had no effect. The UGM was also found to be essential for supporting the growth of small glandular elements derived from the ventral prostate of pubescent rats. Segments isolated from the terminal vesicles (TIPs) and from prostatic tissue adjacent to the urethra (PDCT) regressed when implanted alone under the kidney capsule. However, combination of the prostatic segments with UGM produced prostatic glands with relative wet weight and DNA content responses of the following order: UGM + TIP greater than UGM + PDCT = UGM + UGE. Two-dimensional gel electrophoretic protein patterns from UGM + PDCT and UGM + TIP specimens had differential expression of three protein regions unique to the ventral prostate Quantitative and qualitative responses of the TIP and PDCT segments to UGM inductive influences indicate that differences exist between the epithelia of the TIP and PDCT regions of the ventral lobes of the rat prostate.     

Cells of origin of the hippocampal afferent projection from the nucleus reuniens thalami

Summary Neurons of the nucleus reuniens thalami stained with Golgi methods are compared to cells in this nucleus labelled in retrograde fashion after hippocampal injections of horseradish peroxidase. The cellular morphology ranges from fusiform to multiangular with most cells showing radiating processes characteristic of neurons in the reticular core. Dendrites are long and relatively smooth, with a few sparsely distributed spinous processes. These cells are comparable to the cholinergic cells of the median septal/diagonal band area which also project into the hippocampal formation.We would like to thank Mr. Al Cibivicious for his excellent technical assistance. This research was supported in part by general research funds awarded to R.H.B. by East Tennessee State University     

Anti-IgD enhancement of primary antibody responses in rats

The role of membrane IgD in immune responses was examined by treating adult rats with anti-IgD. Anti-IgD when administered to rats in conjunction with optimal or suboptimal doses of either SRBC, a T-dependent antigen, or DNP-Ficoll, a T-independent antigen, enhanced the antibody responses. The greatest enhancement was obtained when anti-IgD was administered before the antigen. The effects of anti-IgD on antibody responses to SRBC were: (i) significant antibody responses to suboptimal antigen concentrations; (ii) greater antibody responses to optimal antigen concentrations; (iii) accelerated antibody responses; (iv) an early shift from IgM to IgG antibodies; (v) prolonged antibody responses. Similar effects on the immune response to DNP-Ficoll were observed with the exception that all antibodies were 2ME sensitive (IgM). These results suggest that an anamnestic type of immune response can be induced in anti-IgD-treated rats when given a primary antigen exposure. Injection of anti-IgD without SRBC or DNP-Ficoll induces B-cell proliferation without detectable antibody production to these antigens, indicating at least two signals are required for the enhanced antibody responses.     

Analysis of a Coxiella burnetti gene product that activates capsule synthesis in Escherichia coli: requirement for the heat shock chaperone DnaK and the two-component regulator RcsC.

A 1.2-kb EcoRI genomic DNA fragment of Coxiella burnetti, when cloned onto a multicopy plasmid, was found to induce capsule synthesis (mucoidy) in Escherichia coli. Nucleotide sequence analysis revealed the presence of an open reading frame that could encode a protein of 270 amino acids. Insertion of a tet cassette into a unique NruI restriction site resulted in the loss of induction of mucoidy. Because of its ability to induce mucoidy, we designated this gene mucZ. Computer search for homologies to mucZ revealed 42% identity to an open reading frame located at 1 min of the E. coli chromosome. Interestingly, the C-terminal amino acid residues of MucZ share significant homology with the J domain of the DnaJ protein and its homologs, suggesting potential interactions between MucZ and components of the DnaK-chaperone machinery. Results presented in this paper suggest that E. coli requires DnaK-chaperone machinery for Lon-RcsA-mediated induction of capsule synthesis, as noticed first by S. Gottesman (personal communication). The induction caused by MucZ is independent of Lon-RcsA and is mediated through the two-component regulators RcsC and RcsB. DnaK and GrpE but not DnaJ are also required for the RcsB-mediated MucZ induction, and we propose that MucZ is a DnaJ-like chaperone protein that might be required for the formation of an active RcsA-RcsB complex and for the RcsC-dependent phosphorylation of RcsB. Discussions are presented that suggest three different roles for alternative forms of the DnaK-chaperone machinery in capsule production.     

Evolution of feline leukemia virus variant genomes with insertions, deletions, and defective envelope genes in infected cats with tumors.

In order to study retroviral variation, selection, and viral correlates of in vivo pathogenicity, we documented the evolution of feline leukemia virus (FeLV) variants in cats that died with thymic lymphoma after infection with molecularly cloned subgroup A FeLV. Using genomic DNA from cat necropsy samples, we employed PCR to amplify and clone the envelope gene, which is a major determinant of the specific pathogenicity of different FeLV variants. In the envelope gene, mutations encoded scattered amino acid changes that did not cluster into clearly definable variable regions; however, characterization of these terminal variant sequences revealed a predominance of G-to-A and A-to-G nucleotide substitutions. Additionally, some cats harbored variants with recombinant subgroup B-like envelope genes, while the major variant from one cat had a 12-bp insertion in a region previously characterized as an immunodeficiency-inducing determinant. Finally, proviruses from tumor DNA frequently possessed envelope genes predicted to encode a protein truncated in the N-terminal half because of either premature termination codons or deletions ranging from 29 to 1,666 bp. In contrast, all envelope genes cloned from the bone marrow of one cat were predicted to encode full-length envelope product, and only a minority of proviral clones from a cat that did not develop a tumor had defective envelope genes. Thus, in the cat, viruses evolved from subgroup A FeLV that had point mutations, insertions, deletions, or recombinant envelope genes. Furthermore, defective variants were particularly prominent in T-cell tumors.