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Identification of Functional mob Regions in Rhizobium etli: Evidence for Self-Transmissibility of the Symbiotic Plasmid pRetCFN42d. Daniel Pérez-Mendoza, 2004.An approach originally designed to identify functional origins of conjugative transfer (oriT or mob) in a bacterial genome (J . A . Herrera-Cervera, J . M . Sanjuán-Pinilla, J . Olivares, and J . Sanjuán, J . Bacteriol . 180:4583-4590, 1998) was modified to improve its reliability and prevent selection of undesired false mob clones . By following this modified approach, we were able to identify two functional mob regions in the genome of Rhizobium etli CFN42 . One corresponds to the recently characterized transfer region of the nonsymbiotic, self-transmissible plasmid pRetCFN42a (C . Tun-Garrido, P . Bustos, V . González, and S . Brom, J . Bacteriol . 185:1681-1692, 2003), whereas the second mob region belongs to the symbiotic plasmid pRetCFN42d . The new transfer region identified contains a putative oriT and a typical conjugative (tra) gene cluster organization . Although pRetCFN42d had not previously been shown to be self-transmissible, mobilization of cosmids containing this tra region required the presence of a wild-type pRetCFN42d in the donor cell; the presence of multiple copies of this mob region in CFN42 also promoted conjugal transfer of the Sym plasmid pRetCFN42d . The overexpression of a small open reading frame, named yp028, located downstream of the putative relaxase gene traA, appeared to be responsible for promoting the conjugal transfer of the R . etli pSym under laboratory conditions . This yp028-dependent conjugal transfer required a wild-type pRetCFN42d traA gene . Our results suggest for the first time that the R . etli symbiotic plasmid is self-transmissible and that its transfer is subject to regulation . In wild-type CFN42, pRetCFN42d tra gene expression appears to be insufficient to promote plasmid transfer under standard laboratory conditions; gene yp028 may play some role in the activation of conjugal transfer in response to as-yet-unknown environmental conditions . Spreading Factors of Mycoplasma alligatoris, a Flesh-Eating Mycoplasma. D. R. Brown, 2004.Mycoplasma alligatoris causes lethal invasive disease of alligators and caimans . A homolog of the nagH gene, encoding a hyaluronidase secreted by Clostridium perfringens, and a C . perfringens hyaluronidase nagI or nagK pseudogene were discovered in the M . alligatoris genome . The nagH gene was detected by PCR in the closest relative of M . alligatoris, Mycoplasma crocodyli, but not in 40 other species representing the Mycoplasma hominis, Mycoplasma pneumoniae, and Spiroplasma phylogenetic clusters . The hyaluronidase activity in the cellular fraction of M . alligatoris and M . crocodyli SP4 broth cultures was equivalent to 10–16 U of Streptomyces hyalurolyticus hyaluronidase CFU–1 . Negligible activity was present in the cell-free supernatant fraction . No chondroitinase activity was detected . There is also a novel homolog of the nanI gene, which encodes a sialidase secreted by C . perfringens, in the M . alligatoris genome . The signature YRIP and SXDXGXTW motifs and catalytic residues of the clostridial sialidase are conserved in the mycoplasmal gene, but the leader sequence necessary for its secretion by C . perfringens is absent . The gene was not detected by PCR in any other mycoplasma . Potent cell-associated sialidase activity was present in M . alligatoris colonies on agar but not in the cell-free supernatants of broth cultures or in M . crocodyli . The presence of hyaluronidase and sialidase in M . alligatoris is consistent with the rapid invasiveness and necrotizing effects of this organism, and the lack of sialidase in M . crocodyli is consistent with its comparatively attenuated virulence . This genetic and biochemical evidence suggests that the spreading factors hyaluronidase and sialidase, a combination unprecedented in mycoplasmas, are the basis of the virulence of M . alligatoris . Potential for Interactions between Caspofungin and Nelfinavir or Rifampin. Julie A. Stone, 2004.The potential for interactions between caspofungin and nelfinavir or rifampin was evaluated in two parallel-panel studies . In study A, healthy subjects received a 14-day course of caspofungin alone (50 mg administered intravenously [IV] once daily) (n = 10) or with nelfinavir (1,250 mg administered orally twice daily) (n = 9) or rifampin (600 mg administered orally once daily) (n = 10) . In study B, 14 subjects received a 28-day course of rifampin (600 mg administered orally once daily), with caspofungin (50 mg administered IV once daily) coadministered on the last 14 days, and 12 subjects received a 14-day course of caspofungin alone (50 mg administered IV once daily) . The coadministration/administration alone geometric mean ratio for the caspofungin area under the time-concentration profile calculated for the 24-h period following dosing [AUC0-24] was as follows (values in parentheses are 90% confidence intervals [CIs]): 1.08 (0.93-1.26) for nelfinavir, 1.12 (0.97-1.30) for rifampin (study A), and 1.01 (0.91-1.11) for rifampin (study B) . The shape of the caspofungin plasma profile was altered by rifampin, resulting in a 14 to 31% reduction in the trough concentration at 24 h after dosing (C24h), consistent with a net induction effect at steady state . Both the AUC and the C24h were elevated in the initial days of rifampin coadministration in study A (61 and 170% elevations, respectively, on day 1) but not in study B, consistent with transient net inhibition prior to full induction . The coadministration/administration alone geometric mean ratio for the rifampin AUC0-24 on day 14 was 1.07 (90% CI, 0.83-1.38) . Nelfinavir does not meaningfully alter caspofungin pharmacokinetics . Rifampin both inhibits and induces caspofungin disposition, resulting in a reduced C24h at steady state . An increase in the caspofungin dose to 70 mg, administered daily, should be considered when the drug is coadministered with rifampin . Novel Pathway for Alcoholic Fermentation of  -Gluconolactone in the Yeast Saccharomyces bulderi.Johannes P. van Dijken, 2002.Under anaerobic conditions, the yeast Saccharomyces bulderi rapidly ferments -gluconolactone to ethanol and carbon dioxide . We propose that a novel pathway for
-gluconolactone fermentation operates in this yeast . In this pathway,
-gluconolactone is first reduced to glucose via an NADPH-dependent glucose dehydrogenase (EC 1.1.1.47) . After phosphorylation, half of the glucose is metabolized via the pentose phosphate pathway, yielding the NADPH required for the glucose-dehydrogenase reaction . The remaining half of the glucose is dissimilated via glycolysis . Involvement of this novel pathway in
-gluconolactone fermentation in S . bulderi is supported by several experimental observations . (i) Fermentation of
-gluconolactone and gluconate occurred only at low pH values, at which a substantial fraction of the substrate is present as
-gluconolactone . Unlike gluconate, the latter compound is a substrate for glucose dehydrogenase . (ii) High activities of an NADP+-dependent glucose dehydrogenase were detected in cell extracts of anaerobic,
-gluconolactone-grown cultures, but activity of this enzyme was not detected in glucose-grown cells . Gluconate kinase activity in cell extracts was negligible . (iii) During anaerobic growth on
-gluconolactone, CO2 production exceeded ethanol production by 35%, indicating that pyruvate decarboxylation was not the sole source of CO2 . (iv) Levels of the pentose phosphate pathway enzymes were 10-fold higher in
-gluconolactone-grown anaerobic cultures than in glucose-grown cultures, consistent with the proposed involvement of this pathway as a primary dissimilatory route in
-gluconolactone metabolism .
Agrobacterium tumefaciens Twin-Arginine-Dependent Translocation Is Important for Virulence, Flagellation, and Chemotaxis but Not Type IV Secretion. Zhiyong Ding, 2003.This study characterized the contribution of the twin-arginine translocation (TAT) pathway to growth, motility, and virulence of the phytopathogen Agrobacterium tumefaciens . In contrast to wild-type strain A348, a tatC null mutant failed to export the green fluorescent protein fused to the trimethylamine N-oxide reductase (TorA) signal sequence or to grow on nitrate as a sole electron acceptor during anaerobic growth . The tatC mutant displayed defects in growth rate and cell division but not in cell viability, and it also released abundant levels of several proteins into the culture supernatant when grown in rich medium or in vir induction minimal medium . Nearly all A348 cells were highly motile in both rich and minimal media . By contrast, approximately 0.1% of the tatC mutant cells were motile in rich medium, and <0.01% were motile in vir induction medium . Nonmotile tatC mutant cells lacked detectable flagella, whereas motile tatC mutant cells collected from the edge of a motility halo possessed flagella but not because of reversion to a functional TAT system . Motile tatC cells failed to exhibit chemotaxis toward sugars under aerobic conditions or towards nitrate under anaerobic conditions . The tatC mutant was highly attenuated for virulence, only occasionally (  15% of inoculations) inciting formation of small tumors on plants after a prolonged incubation period of 6 to 8 weeks . However, an enriched subpopulation of motile tatC mutants exhibited enhanced virulence compared to the nonmotile variants . Finally, the tatC mutant transferred T-DNA and protein effectors to plant cells and a mobilizable IncQ plasmid to agrobacterial recipients at wild-type levels . Together, our findings establish that, in addition to its role in secretion of folded cofactor-bound enzymes functioning in alternative respiration, the TAT system of A . tumefaciens is an important virulence determinant . Furthermore, this secretion pathway contributes to flagellar biogenesis and chemotactic responses but not to sensory perception of plant signals or the assembly of a type IV secretion system .
Antifungal Activity of Phenyllactic Acid against Molds Isolated from Bakery Products. Paola Lavermicocca, 2003.Phenyllactic acid (PLA) has recently been found in cultures of Lactobacillus plantarum that show antifungal activity in sourdough breads . The fungicidal activity of PLA and growth inhibition by PLA were evaluated by using a microdilution test and 23 fungal strains belonging to 14 species of Aspergillus, Penicillium, and Fusarium that were isolated from bakery products, flours, or cereals . Less than 7.5 mg of PLA ml-1 was required to obtain 90% growth inhibition for all strains, while fungicidal activity against 19 strains was shown by PLA at levels of  10 mg ml-1 . Levels of growth inhibition of 50 to 92.4% were observed for all fungal strains after incubation for 3 days in the presence of 7.5 mg of PLA ml-1 in buffered medium at pH 4, which is a condition more similar to those in real food systems . Under these experimental conditions PLA caused an unpredictable delaying effect that was more than 2 days long for 12 strains, including some mycotoxigenic strains of Penicillium verrucosum and Penicillium citrinum and a strain of Penicillium roqueforti (the most widespread contaminant of bakery products); a growth delay of about 2 days was observed for seven other strains . The effect of pH on the inhibitory activity of PLA and the combined effects of the major organic acids produced by lactic acid bacteria isolated from sourdough bread (PLA, lactic acid, and acetic acid) were also investigated . The ability of PLA to act as a fungicide and delay the growth of a variety of fungal contaminants provides new perspectives for possibly using this natural antimicrobial compound to control fungal contaminants and extend the shelf lives of foods and/or feedstuffs .
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