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Broth Microdilution Susceptibility Testing for Leptospira spp.. Clinton K. Murray, 2004.Leptospirosis in humans has traditionally been treated with penicillin or doxycycline . The choice of therapy offered at the time of initial patient presentation is often empirical, as definitive diagnosis can take weeks . Determining the activity of numerous antimicrobial agents against a wide range of Leptospira serovars may broaden empirical therapeutic options . Various antimicrobials have been shown to be active against a limited number of serovars in in vitro studies, chiefly by the use of broth macrodilution techniques . We developed a broth microdilution technique using the commercially available growth indicator alamarBlue . MICs produced by this technique were compared to MICs and minimal bactericidal concentrations produced by the traditional broth macrodilution technique . The internal validity of our methods was assessed with 11 runs over numerous days with a single isolate of Leptospira interrogans serovar Icterohaemorrhagiae . By either method, the MICs for these internal-validity runs fell within 2 dilutions of each other for more than 90% of antimicrobials . A broader application of these two techniques included 12 serovars (including seven species) of Leptospira and six antimicrobials (penicillin G, doxycycline, chloramphenicol, erythromycin, cefotaxime, and ciprofloxacin) . Observed reproducibility fell within 2 dilutions for 99% of the duplicate result sets for the MIC microdilution method, compared to 89% for the MIC macrodilution method . The macrodilution method tended to have a higher MIC at which 90% of the isolates were inhibited (MIC90) than did the microdilution method, but the MIC90s of both methods were within 2 dilutions of each other for all six drugs . The macrodilution and microdilution techniques produced similar results, with microdilution allowing a faster, more streamlined method of producing MIC results . Display of Bacterial Lipase on the Escherichia coli Cell Surface by Using FadL as an Anchoring Motif and Use of the Enzyme in Enantioselective Biocatalysis. Seung Hwan Lee, 2004.We have developed a novel cell surface display system by employing FadL as an anchoring motif, which is an outer membrane protein involved in long-chain fatty acid transport in Escherichia coli . A thermostable Bacillus sp . strain TG43 lipase (44.5 kDa) could be successfully displayed on the cell surface of E . coli in an active form by C-terminal deletion-fusion of lipase at the ninth external loop of FadL . The localization of the truncated FadL-lipase fusion protein on the cell surface was confirmed by confocal microscopy and Western blot analysis . Lipase activity was mainly detected with whole cells, but not with the culture supernatant, suggesting that cell lysis was not a problem . The activity of cell surface-displayed lipase was examined at different temperatures and pHs and was found to be the highest at 50°C and pH 9 to 10 . Cell surface-displayed lipase was quite stable, even at 60 and 70°C, and retained over 90% of the full activity after incubation at 50°C for a week . As a potential application, cell surface-displayed lipase was used as a whole-cell catalyst for kinetic resolution of racemic methyl mandelate . In 36 h of reaction, (S)-mandelic acid could be produced with the enantiomeric excess of 99% and the enantiomeric ratio of 292, which are remarkably higher than values obtained with crude lipase or cross-linked lipase crystal . These results suggest that FadL may be a useful anchoring motif for displaying enzymes on the cell surface of E . coli for whole-cell biocatalysis . Use of a Promoter Trap To Identify Bacillus cereus Genes Regulated by Tomato Seed Exudate and a Rhizosphere Resident, Pseudomonas aureofaciens. Anne K. Dunn, 2003.The goal of this study was to identify genes in Bacillus cereus, a bacterium commonly associated with plant seeds and roots, that are affected by compounds originating from a host plant, tomato, or another rhizosphere resident, Pseudomonas aureofaciens . We constructed a B . cereus chromosomal DNA library in a promoter-trap plasmid, pAD123, which contains a promoterless version of the green fluorescent protein (GFP) gene, gfpmut3a . The library was screened by using fluorescence-activated cell sorting for clones showing a change in GFP expression in response to either tomato seed exudate or culture supernatant of P . aureofaciens strain 30-84 . We identified two clones carrying genes that were induced by the presence of tomato seed exudate and nine clones carrying genes that were repressed by P . aureofaciens culture supernatant . A clone chosen for further study contained an open reading frame, designated lipA, that encodes a deduced protein with a lipoprotein signal peptide sequence similar to lipoproteins in B . subtilis. Expression of gusA under control of the lipA promoter increased twofold when cells were exposed to tomato seed exudate and in a concentration-dependent manner when exposed to a mixture of amino acids . When the wild type and a 10-fold excess of a lipA mutant were applied together to tomato seeds, 2 days after planting, the wild type displayed medium-dependent culturability, whereas the lipA mutant was unaffected . This study demonstrates the power of a promoter trap to identify genes in a gram-positive bacterium that are regulated by the biotic environment and resulted in the discovery of lipA, a plant-regulated gene in B . cereus . High-Resolution Differentiation of Cyanobacteria by Using rRNA-Internal Transcribed Spacer Denaturing Gradient Gel Electrophoresis. Ingmar Janse, 2003.For many ecological studies of cyanobacteria, it is essential that closely related species or strains can be discriminated . Since this is often not possible by using morphological features, cyanobacteria are frequently studied by using DNA-based methods . A powerful method for analysis of the diversity and dynamics of microbial populations and for checking the purity and affiliation of cultivated strains is denaturing gradient gel electrophoresis (DGGE) . We realized high-resolution discrimination of a variety of cyanobacteria by means of DGGE analysis of sections of the internal transcribed spacer between the 16S and 23S rRNA genes (rRNA-ITS) . A forward primer specific for cyanobacteria, targeted at the 3' end of the 16S rRNA gene, was designed . The combination of this primer and three different reverse primers targeted to the rRNA-ITS or to the 23S rRNA gene yielded PCR products of different sizes from cultures of all 16 cyanobacterial genera that were tested but not from other bacteria . DGGE profiles produced from the shortest section of rRNA-ITS consisted of one band for all but one cyanobacterial genera, and those generated from longer stretches of rRNA-ITS yielded DGGE profiles containing one to four bands . The suitability of DGGE for detecting intrageneric and intraspecific variation was tested by using strains of the genus Microcystis . Many strains could be discriminated by means of rRNA-ITS DGGE, and the resolution of this method was strikingly higher than that obtained with previously described methods . The applicability of the developed DGGE assays for analysis of cyanobacteria in field samples was demonstrated by using samples from freshwater lakes . The advantages and disadvantages associated with the use of each developed primer set are discussed .
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