Identification and Cloning of Genes Involved in Specific Desulfurization of Dibenzothiophene by Rhodococcus sp. Strain IGTS8

The gram-positive bacterium Rhodococcus sp. strain IGTS8 is able to remove sulfur from certain aromatic compounds without breaking carbon-carbon bonds. In particular, sulfur is removed from dibenzothiophene (DBT) to give the final product, 2-hydroxybiphenyl. A genomic library of IGTS8 was constructed in the cosmid vector pLAFR5, but no desulfurization phenotype was imparted to Escherichia coli. Therefore, IGTS8 was mutagenized, and a new strain (UV1) was selected that had lost the ability to desulfurize DBT. The genomic library was transferred into UV1, and several colonies that had regained the desulfurization phenotype were isolated, though free plasmid could not be isolated. Instead, vector DNA had integrated into either the chromosome or a large resident plasmid. DNA on either side of the inserted vector sequences was cloned and used to probe the original genomic library in E. coli. This procedure identified individual cosmid clones that, when electroporated into strain UV1, restored desulfurization. When the origin of replication from a Rhodococcus plasmid was inserted, the efficiency with which these clones transformed UV1 increased 20- to 50-fold and they could be retrieved as free plasmids. Restriction mapping and subcloning indicated that the desulfurization genes reside on a 4.0-kb DNA fragment. Finally, the phenotype was transferred to Rhodococcus fascians D188-5, a species normally incapable of desulfurizing DBT. The mutant strain, UV1, and R. fascians produced 2-hydroxybiphenyl from DBT when they contained appropriate clones, indicating that the genes for the entire pathway have been isolated.     

Histidine substitution identifies a surface position and confers Cs+ selectivity on a K+ pore.

The amino acid located at position 369 is a key determinant of the ion conduction pathway or pore of the voltage-gated K+ channels, Kv2.1 and a chimeric channel, CHM, constructed by replacing the pore region of Kv2.1 with that of Kv3.1. To determine the orientation of residue 369 with respect to the aqueous lumen of the pore, the nonpolar Ile at 369 in Kv2.1 was replaced with a basic His. This substitution produced a Cs(+)-selective channel with Cs+:K+ permeability ratio of 4 compared to 0.1 in the wild type. Block by external tetraethylammonium (TEA) was reduced about 20-fold, while block by internal TEA was unaffected. External protons and Zn2+, that are known to interact with the imidazole ring of His, blocked the mutant channel much more effectively than the wild type channel. The blockade by Zn2+ and protons was voltage-independent, and the proton blockade had a pKa of about 6.5, consistent with the pKa for His in solution. The histidyl-specific reagent diethylpyrocarbonate produced greatly exaggerated blockade of the mutated channel compared to the wild type. The residue at position 369 appears to form part of the binding site for external TEA and to influence the selectivity for monovalent cations. We suggest that the imidazole side-chain of His369 is exposed to the aqueous lumen at a surface position near the external mouth of the pore.     

Exotoxin production by Aeromonas spp. in foods

A psychrotrophic toxin-producing strain of Aeromonas hydrophila grew well in a range of food slurries (scallop, prawn, fish, chicken liver paté, liverwurst, chicken luncheon slice and commercial baby food preparations) held at refrigeration temperatures. In most foods, excluding the baby food preparations, exotoxins were produced at levels comparable with production in bacteriological broth without apparent food spoilage (all but prawn and fish). Addition of ultra-heat treated (UHT) milk to toxin-containing broth culture supernatants markedly decreased or removed haemolytic and cytotoxic activities, explaining low levels of toxins found in milk in a previous study. Baby food preparations did not inactivate exotoxins under similar conditions suggesting production of toxins rather than their inactivation was inhibited in these foods.     

DNA/DNA hybridization studies of carnivorous marsupials. III. Relationships among species ofDidelphis (Didelphidae)

We report three sets of DNA hybridization experiments conducted to determine relationships among species ofDidelphis (D. albiventris, D. marsupialis, D. virginiana). The 1989 and 1991 sets had fewer replicates per cell than the 1990 series (3.4 and 5.4 vs 9), but in 1991 we distinguished two populations ofD. marsupialis and utilized several individuals for each heterologous comparison. BothPhilander opossum andLutreolina crassicaudata were used as outgroups in 1989, but onlyLutreolina was included in subsequent sets. For each set, we calculated all four standard indices of thermal stability (T _mode,T _m,T ₅₀ H, and NPH) and constructed trees by least-squares (FITCH) and neighbor-joining methods, both before and after correction for asymmetric reciprocal cell values. Subsets of the 1989 data lacking eitherPhilander orLutreolina were analyzed similarly. To explore measurement imprecision, the corrected and uncorrected matrices for each of the four indices were bootstrapped 100 times for the 1989 set and subsets and 1000 times for the 1990 and 1991 sets. Again, for the 1990 and 1991 data, an additional 100 bootstrapped distances were fitted to user trees representing the three possible pairings ofDidelphis spp. to determine the significance of the FITCH branch lengths. The successive experimental sets generated increasingly consistent evidence for pairingD. marsupialis withD. albiventris. The 1989 experiments involved just 85 comparisons, and only T _mode's pairedD. marsupialis withD. albiventris (at bootstrap percentages of 70% or above), but did so whetherPhilander orLutreolina or both were included as the outgroup(s). FITCH and neighbor-joining trees had identical topologies for T _mode's but sometimes differed for the other measures. In contrast, all but one of the corresponding FITCH and neighborjoining trees matched for the 1990 and 1991 data, and three of the four distance measures (T _mode, T _m, and T ₅₀ H) unitedD. marsupialis withD. albiventris at bootstrap percentages averaging 81%; NPH gave a different result for 1990, associatingD. marsupialis withD. virginiana. Further, all but 2 of the 16 matrices for 1990 and 1991 gave mean bootstrapped branch lengths for a consensus pairing that were positive and at least one standard deviation from zero, despite the very short internodes recovered. These results illustrate that the potential of DNA hybridization for resolving very close relationships depends on both the index and the experimental design employed. We conclude that of the three species,D. albiventris andD. marsupialis shared a more recent common ancestor and estimate thatDidelphis spp. have diverged at about 0.39% in nucleotide sequence per myr.Deceased.     

A molecular perspective on the phylogeny of placental mammals based on mitochondrial 12S rDNA sequences,with special reference to the problem of the Paenungulata

Part of the 12S rDNA gene was amplified and sequenced for 11 placental mammals, 3 marsupials, and 2 monotremes. Multiple alignments for these sequences and nine additional placental sequences taken from GenBank were obtained using CLUSTAL. Phylogenetic analyses were performed using standard parsimony, transversion parsimony, and Lake's method of invariants. All of our analyses uniteLoxodontia withDugong. Procavia, in turn, is a sister group to these taxa, thus supporting the monophyly of the Paenungulata. Perissodactyls are a sister group to paenungulates when transitions and transversions are both included but not when transitions are omitted. Likewise, cetaceans are a sister group to artiodactyls on minimum length trees under standard parsimony but not under transversion parsimony. Rodent monophyly and bat monophyly also receive mixed support, as does a putative alliance between primates and lagomorphs. Interestingly, the percentage divergence between the echidna and the platypus is less than for the rat and mouse.     

Seasonal dynamics of the association between sweet potato and vesicular-arbuscular mycorrhizal fungi

To better understand the behavior of selected vesicular-arbuscular mycorrhizal (VAM) isolates in the field, we documented the growth of roots, root hairs, and VAM colonization of inoculated and noninoculated sweet potato plants (Ipomea batatas (L.) Lam. cv White Star) over a growing season. We also determined the seasonal dynamics of P and Zn uptake, and shoot and storage-root growth. Shoot cuttings were inoculated with an isolate of either Glomus etunicatum Becker and Gerdemann or Acaulospora rugosa Mortan, or were not inoculated, and were harvested 2, 4, 8, 13, 20, and 27 weeks after planting (WAP). At each harvest, roots were sampled at 0 to 30, 30 to 60, and 60 to 90 cm depths and at 0, 23, 83, and 116 cm from the base of the shoot. At the end of the study, the roots of three non-inoculated plants were sampled by soil horizon. Inoculation had no affect on shoot growth or total shoot uptake of P and Zn; shoot dry mass and P and Z content increased rapidly up to 20 WAP, while shoot length continued to increase through 27 WAP. Shoot-P concentration of plants inoculated with A. rugosa at 2 and 8 WAP were higher than the noninoculated plants, while shoot-Zn concentration was not affected by inoculation. Storage-root yields of inoculated plants were higher than yields for noninoculated plants. Root length density, and percentage of root length with root hairs and VAM colonization were highest and most dynamic near the base of the plant. Percentage of root length colonization by VAM fungi was highest in the E2 horizon, intermediate in the Bh horizon, and lowest in the Ap horizon. Percentage of root length with root hairs had the opposite pattern. Intensive measurements of root characteristics close to the base of the plant, and shoot P-content and concentration during the period of rapid yield production, provided the most useful data for evaluating the activity of effective isolates.Published as Florida Agricultural Experimental Station Journal Series No. R-02576     

Localization of collagens and alkaline phosphatase activity during mineralization and ossification of human first rib cartilage

The localization of type X collagen and alkaline phosphatase activity was examined in order to gain a better understanding of tissue remodelling during development of human first rib cartilage. First rib cartilages from children and adolescents showed no staining for type X collagen and alkaline phosphatase activity. After onset of mineralization in the late second decade, a peripheral ossification process preceded by mineralized fibrocartilage could be distinguished from a more central one preceded by mineralized hyaline cartilage. No immunostaining for type X collagen was found in either type of cartilage. However, strong staining for alkaline phosphatase activity was detected around chondrocyte-like cells within fibrocartilage adjacent to the peripheral mineralization front, while a weaker staining pattern was observed around chondrocytes of hyaline cartilage near the central mineralization front. In addition, the territorial matrix of some chondrocytes within the hyaline cartilage revealed staining for type I collagen, suggesting that these cells undergo a dedifferentiation process, which leads to a switch from type II to type I collagen synthesis. The study provides evidence that mineralization of the hyaline cartilage areas in human first rib cartilage occurs in the absence of type X collagen synthesis but in the presence of alkaline phosphatase. Thus, mineralization of first rib cartilage seems to follow a different pattern from endochondral ossification in epiphyseal discs.     

Gap junctional intercellular communication of bovine luteal cells from several stages of the estrous cycle: Effects of prostaglandin F2α, protein kinase C and calcium

Cellular interactions mediated by both contact-dependent and contact-independent mechanisms are probably important to maintain luteal function. The present studies were performed to evaluate the effects of luteotropic and luteolytic hormones, and also intracellular regulators, on contact-dependent gap junctional intercellular communication (GJIC) of bovine luteal cells from several stages of luteal development. Bovine corpora lutea (CL) from the early, mid and late luteal phases of the estrous cycle were dispersed with collagenase and incubated with no treatment, LH, PGF or LH + PGF (Experiment 1), or with no treatment, or agonists or antagonists of protein kinase C (TPA or H-7) or calcium (A23187 or EGTA; Experiment 2). After incubation, media were collected for determination of progesterone concentrations. Then the rate of GJIC was evaluated for small luteal cells in contact with small luteal cells, and large luteal cells in contact with small luteal cells by using the fluorescence recovery after photobleaching technique and laser cytometry. Luteal cells from each stage of the estrous cycle exhibited GJIC, but the rate of GJIC was least (P<0.05) for luteal cells from the late luteal phase. LH increased (P<0.05) GJIC between small luteal cells from the mid and late but not the early luteal phase. PGF increased (P<0.05) GjIC between small luteal cells from the mid luteal phase and diminished (P<0.05) LH-stimulatory effects on GjIC between small luteal cells from the late luteal phase. Throughout the estrous cycle, TPA decreased (P<0.05) the rate of GjIC between large and small, and between small luteal cells, and A23187 decreased (P<0.05) the rate of GJIC between large and small luteal cells. LH and LH + PGF, but not PGF alone increased (P<0.05) progesterone secretion by luteal cells from the mid and late luteal phases. Agonists or antagonists of PKC or calcium did not affect progesterone secretion by luteal cells. These data demonstrate that both luteal cell types communicate with small luteal cells, and the rate of communication depends on the stage of luteal development. LH and PGF affect GjIC between small luteal cells during the fully differentiated (mid-luteal) and regressing (late luteal) stages of the estrous cycle. In contrast, at all stages of luteal development, activation of PKC decreases GjIC between small and between large and small luteal cells, whereas calcium ionophore decreases GjIC only between large and small luteal cells. Luteotropic and luteolytic hormones, and intracellular regulators, may be involved in regulation of cellular interactions within bovine CL which likely is an important mechanism for coordination of luteal function.     

Isolation and characteristics of a wheatbran-degrading Butyrivibrio from human faeces

Screening over 100 isolates from human faeces for cellulolytic activity led to the isolation of a weakly cellulolytic anaerobic, curved, motile bacterium which produced H₂, lactate and butyrate from wheatbran. The mol% of G + C in the DNA was 39–42. These properties, together with the Gram-positive cell wall ultrastructure and SDS-PAGE profile, are consistent with the genus Butyrivibrio. The isolate is believed to be the most active wheatbran-degrading bacterium so far described.     

Multiple residues specify external tetraethylammonium blockade in voltage-gated potassium channels.

We have mapped residues in the carboxyl half of the P region of a voltage-gated K+ channel that influence external tetraethylammonium (TEA) block. Fifteen amino acids were substituted with cysteine and expressed in oocytes from monomeric or heterodimeric cRNAs. From a total of six mutant channels with altered TEA sensitivity, three were susceptible to modification by extracellularly applied charged methanethiosulfonates (MTSX). Another residue did not affect TEA block but was protected from MTSX by TEA. MTSX modification of position Y380C, thought to form the TEA binding site, affected TEA affinity only moderately, and this effect could be reversed by additional charge transfer from an oppositely charged MTSX analog. The results show that TEA block is modulated from multiple sites, including residues located deep in the pore and that several side chains besides that of Y380 are exposed at the TEA receptor.