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Targeted Mutagenesis of the Mycobacterium smegmatis mca Gene, Encoding a Mycothiol-Dependent Detoxification Protein. Mamta Rawat, 2004.Mycothiol [MSH], a functional analogue of glutathione [GSH]that is found exclusively in actinomycetes, reacts with electrophilesand toxins to form MSH-toxin conjugates . Mycothiol S-conjugate amidase [Mca] then catalyzes the hydrolysis of an amide bondin the S conjugates, producing a mercapturic acid of the toxin,which is excreted from the bacterium, and glucosaminyl inositol,which is recycled back to MSH . In this study, we have generatedand characterized an allelic exchange mutant of the mca geneof Mycobacterium smegmatis . The mca mutant accumulates the S conjugates of the thiol-specific alkylating agent monobromobimane and the antibiotic rifamycin S . Introduction of M . tuberculosis mca epichromosomally or introduction of M . smegmatis mca integrativelyresulted in complementation of Mca activity and reduced levelsof S conjugates . The mutation in mca renders the mutant strainmore susceptible to electrophilic toxins, such as N-ethylmalemide, iodoacetamide, and chlorodinitrobenzene, and to several oxidants, such as menadione and plumbagin . Additionally we have shown that the mca mutant is also more susceptible to the antituberculous antibiotic streptomycin . Mutants disrupted in genes belonging to MSH biosynthesis are also more susceptible to streptomycin, providing further evidence that Mca detoxifies streptomycinin the mycobacterial cell in an MSH-dependent manner. Inactivation of the Flagellin Gene flaA in Magnetospirillum gryphiswaldense Results in Nonmagnetotactic Mutants Lacking Flagellar Filaments. Daniel Schultheiss, 2004.Magnetotactic bacteria synthesize magnetosomes, which cause them to orient and migrate along magnetic field lines . The analysis of magnetotaxis and magnetosome biomineralization at the molecular level has been hindered by the unavailability of genetic methods, namely the lack of a means to introduce directed gene-specific mutations . Here we report a method for knockout mutagenesis by homologous recombination in Magnetospirillum gryphiswaldense . Multiple flagellin genes, which are unlinked in the genome, were identified in M . gryphiswaldense . The targeted disruption of the flagellin gene flaA was shown to eliminate flagella formation, motility, and magnetotaxis . The techniques described in this paper will make it possible to take full advantage of the forthcoming genome sequences of M . gryphiswaldense and other magnetotactic bacteria . Truncation Analysis of TatA and TatB Defines the Minimal Functional Units Required for Protein Translocation. Philip A. Lee, 2002.The TatA and TatB proteins are essential components of the twin arginine protein translocation pathway in Escherichia coli . C-terminal truncation analysis of the TatA protein revealed that a plasmid-expressed TatA protein shortened by 40 amino acids is still fully competent to support protein translocation . Similar truncation analysis of TatB indicated that the final 30 residues of TatB are dispensable for function . Further deletion experiments with TatB indicated that removal of even 70 residues from its C terminus still allowed significant transport . These results imply that the transmembrane and amphipathic helical regions of TatA and TatB are critical for their function but that the C-terminal domains are not essential for Tat transport activity . A chimeric protein comprising the N-terminal region of TatA fused to the amphipathic and C-terminal domains of TatB supports a low level of Tat activity in a strain in which the wild-type copy of either tatA or tatB (but not both) is deleted . Genetic Organization of the Vibrio harveyi dnaA Gene Region and Analysis of the Function of the V . harveyi DnaA Protein in Escherichia coli. Dvora Berenstein, 2002.The Vibrionaceae family is distantly related to Enterobacteriaceae within the group of bacteria possessing the Dam methylase system . We have cloned, sequenced, and analyzed the dnaA gene region of Vibrio harveyi and found that although the organization of the V . harveyi dnaA region differs from that of Escherichia coli, the expression of both genes is autoregulated and ATP-DnaA binds cooperatively to ATP-DnaA boxes in the dnaA promoter region . The DnaA proteins of V . harveyi and E . coli are interchangeable and function nearly identically in controlling dnaA transcription and the initiation of chromosomal DNA replication despite the evolutionary distance between these bacteria . Molecular Characterization of the Mg2+-Responsive PhoP-PhoQ Regulon in Salmonella enterica. Sergio Lejona, 2003.The PhoP/PhoQ two-component system controls the extracellular magnesium deprivation response in Salmonella enterica . In addition, several virulence-associated genes that are mainly required for intramacrophage survival during the infection process are under the control of its transcriptional regulation . Despite shared Mg2+ modulation of the expression of the PhoP-activated genes, no consensus sequence common to all of them could be detected in their promoter regions . We have investigated the transcriptional regulation and the interaction of the response regulator PhoP with the promoter regions of the PhoP-activated loci phoPQ, mgtA, slyB, pmrD, pcgL, phoN, pagC, and mgtCB . A direct repeat of the heptanucleotide sequence (G/T)GTTTA(A/T) was identified as the conserved motif recognized by PhoP to directly control the gene expression of the first five loci, among which the first four are ancestral to enterobacteria . On the other hand, no direct interaction of the response regulator with the promoter of phoN, pagC, or mgtCB was apparent by either in vitro or in vivo assays . These loci are Salmonella specific and were probably acquired by horizontal DNA transfer . Besides, sequence analysis of pag promoters revealed the presence of a conserved PhoP box in 6 out of the 12 genes analyzed . Our results strongly suggest that the expression of a set of Mg2+-controlled genes is driven by PhoP via unknown intermediate regulatory mechanisms that could also involve ancillary factors . The fdxA Ferredoxin Gene Can Down-Regulate frxA Nitroreductase Gene Expression and Is Essential in Many Strains of Helicobacter pylori. Asish K. Mukhopadhyay, 2003.Very few examples of metabolic regulation are known in the gastric pathogen Helicobacter pylori . An unanticipated case was suggested, however, upon finding two types of metronidazole (Mtz)-susceptible strains: type I, in which frxA (which encodes a nitroreductase that contributes to Mtz susceptibility) is quiescent, and type II, in which frxA is well expressed . Here we report that inactivation of the fdxA ferredoxin gene (hp277) in type I strains resulted in high-level frxA expression (in effect, making them type II) . However, fdxA null derivatives were obtained from only 6 of 32 type I strains tested that were readily transformed with an frxA::aphA marker . This suggested that fdxA is often essential . This essentiality was overcome in 4 of 20 strains by inactivating frxA, which suggested both that frxA overexpression is potentially deleterious and also that fdxA has additional, often vital roles . With type II strains, in contrast, fdxA null derivatives were obtained in 20 of 23 cases tested . Thus, fdxA is dispensable in most strains that normally exhibit (and tolerate) strong frxA expression . We propose that restraint of frxA expression helps maintain balanced metabolic networks in most type I strains, that other homeostatic mechanisms predominate in type II strains, and that these complex results constitute a phenotypic manifestation of H . pylori's great genetic diversity . Detection of Frequency Resonance Energy Transfer Pair on Double-Labeled Microsphere and Bacillus anthracis Spores by Flow Cytometry. E. Zahavy, 2003.Development of an ultrasensitive biosensor for biological hazards in the environment is a major need for pollutant control and for the detection of biological warfare . Fluorescence methods combined with immunodiagnostic methods are the most common . To minimize background noise, arising from the unspecific adsorption effect, we have adapted the FRET (frequency resonance energy transfer) effect to the immunofluorescence method . FRET will increase the selectivity of the diagnosis process by introducing a requirement for two different reporter molecules that have to label the antigen surface at a distance that will enable FRET . Utilizing the multiparameter capability of flow cytometry analysis to analyze the double-labeling/FRET immunostaining will lead to a highly selective and sensitive diagnostic method . This work examined the FRET interaction of fluorescence-labeled avidin molecules on biotin-coated microspheres as a model system . As target system, we have used labeled polyclonal antibodies on Bacillus anthracis spores . The antibodies used were purified immunoglobulin G (IgG) molecules raised in rabbits against B . anthracis exosoporium components . The antibodies were fluorescence labeled by a donor-acceptor chromophore pair, alexa488 as a donor and alexa594 as an acceptor . On labeling the spores with alexa488-IgG as a donor and alexa594-IgG as an acceptor, excitation at 488 nm results in quenching of the alexa-488 fluorescence (Eq = 35%) and appearance of the alexa594 fluorescence (Es = 22%), as detected by flow cytometry analysis . The FRET effect leads to a further isolated gate (FL1/FL3) for the target spores compared to competitive spores such as B . thuringiensis subsp . israelensis and B . subtilis. This new approach, combining FRET labeling and flow cytometry analysis, improved the selectivity of the B . anthracis spores by a factor of 10 with respect to B . thuringiensis subsp . israelensis and a factor of 100 with respect to B . subtilis as control spores .
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