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Mycobacterium abscessus is a rapidly growing mycobacterial species that can be involved in pulmonary and disseminated infections in immunosuppressed or young cystic fibrosis patients. It is an emerging pathogen and has attracted recent attention due to the numerous cases of infection; furthermore, genomic tools have been developed for this species. Nevertheless, the study of this species has until now been limited to spontaneous variants. We report here a comparison of three different mutagenesis systems—the ts-sacB, the phage, and the recombineering systems—and show that there are important differences in their efficiency for the construction of allelic-exchange mutants. We show, using the mmpL4b gene of the glycopeptidolipid pathway as a target, that allelic-exchange mutants can be constructed with a reasonable efficiency (∼7%) using the recombineering system. These observations will facilitate genetic and cellular microbiology experiments involving the construction and use of well-defined mutants to study the virulence determinant of this emerging pathogen.The mycobacterial genus contains plethora of species that are pathogenic for either humans or animals. The most well-known are undoubtedly Mycobacterium leprae, M. tuberculosis, and M. ulcerans, the etiologic agents of human leprosy, tuberculosis, and Buruli ulcer, respectively (47-49). M. avium subsp. paratuberculosis, responsible for Johnes disease in ruminants, is also a serious health concern since it is suspected to be a threat to human via infected milk (9, 10). M. abscessus is an emerging pathogen involved in pulmonary and disseminated infection in young cystic fibrosis patients (26, 36). M. abscessus can cause nosocomial infections of skin and soft tissues in immunosuppressed patients (28, 35). It is also able to cross the blood-brain barrier and to cause meningoencephalitis (42). M. abscessus is phylogenetically related to M. chelonae and, indeed, these species have long been grouped together under the designation of the “M. abscessus-chelonae complex” (6). M. abscessus is a rapid grower that forms colonies in 5 days. Like other mycobacterial species, M. abscessus is equipped with a robust waxy cell wall that, as in other species, probably contributes to virulence (12). The emerging and growing interest in M. abscessus has led to its genome being sequenced (accession no. NC010397) (F. Ripoll et al., unpublished data) and to the development of DNA microarrays (Jean-Yves Coppée, unpublished data).The availability of genomic resources and animal models (32) makes M. abscessus a very attractive system. However, there is no defined mutagenesis system for this species and, to the best of our knowledge, no defined mutants have been constructed thus far. The consequence is that the study of this organism has been restricted to spontaneous variants. Utilization of spontaneous mutants has, nevertheless, allowed the characterization of morphotypically rough isolates that are hypervirulent both in vitro and in vivo (7, 8, 17). These rough isolates are low glycopeptidolipid producers. Glycopeptidolipid is an extractable lipid found at the surface of the bacilli (4, 11, 13). However, its role in the virulence process is currently unknown. The lack of a suitable genetic system is certainly responsible for the rarity of studies on this species (fewer than 500 references in Medline, whereas there are more than 32,000 for M. tuberculosis). Other mycobacterial species, especially M. tuberculosis, have been genetically intractable for many years (15, 18, 24). This has forced researchers to develop dedicated systems for the construction of allelic-exchange mutants. Three major systems have mainly been used thus far in M. tuberculosis and in other mycobacteria: (i) a thermosensitive counterselectable plasmid based on sucrose sensitivity (21-23), (ii) a thermosensitive mycobacteriophage (2) and, more recently, (iii) a mycobacterial recombinase-based system (43, 44). These three systems are effective in M. tuberculosis, M. smegmatis, and other refractory species, including M. avium subsp. avium, and allow straightforward construction of both marked and unmarked mutants.The aim of the present study was to compare the three main mutagenesis systems available for mycobacteria and to determine which system is best adapted to M. abscessus. To this end, we used mmpL4b as a target gene and the three genetic tools described above. The mmpL4b gene is involved in glycopeptidolipid synthesis (29, 40) and is a good model target because its mutation results in a rough phenotype that can be visually distinguished. We show here that there are large differences in efficacy between the three systems and that the mycobacterial recombinase-based system is the most efficient. For an unknown reason, allelic exchange is much less frequent in M. abscessus than in other species, including M. tuberculosis; this complicates the construction of defined mutants. The availability of a suitable genetic system will undoubtedly facilitates the characterization of the virulence determinants in this emerging pathogen.  相似文献   
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Microbially influenced corrosion (MIC) is catalysed by a series of metabolic activities of selected micro-organisms, notably by oxidation of cathodic hydrogen by hydrogenase, by hydrogen sulphide and by reduction of ferric iron. The sulphate-reducing bacteria are considered to be the most common catalyst of MIC, whereas the role of other bacteria has been neglected. This study examined the corrosive potential of the facultative sulphide producer, Shewanella putrefaciens , isolated from an industrial cooling water system. Shewanella putrefaciens was shown to reduce ferric iron and sulphite under anaerobic conditions and with ferric iron being the preferred electron acceptor. The isolate could utilize cathodic hydrogen as an energy source, especially when using sulphite as a terminal electron acceptor. In pure culture corrosion experiments, the highest mass loss of mild steel was observed in the presence of sulphite as sole electron acceptor, although mass loss was also detected where ferric iron was the sole electron acceptor. Our data indicate that S. putefaciens plays a role in MIC as it was able to catalyse a variety of corrosion-promoting reactions and to corrode mild steel under pure culture conditions.  相似文献   
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The present study evaluated the physiological and biochemical mechanisms through which exogenous sodium hydrosulfide (H2S donor) mitigates chromium (Cr) stress in cauliflower. The different levels of Cr included 0, 10, 100 and 200 µM. Results reported that Cr exposure reduced growth and biomass, chlorophyll (Chl) contents, gas exchange parameters and enzymatic antioxidants. Chromium stress enhanced the production of electrolyte leakage (EL), hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents and increased Cr content in the roots, stem, leaf and flowers. Exogenous H2S improved the physiological and biochemical attributes of Cr-stressed cauliflower. Hydrogen sulfide decreased Cr content in different parts of Cr-stressed plants, whereas it increased the Chl contents and gas exchange attributes. H2S reduced the EL, H2O2 and MDA concentrations, enhancing the antioxidant enzymes activities in Cr-stressed roots and leaves compared to the Cr treatments alone. Collectively, our results provide an insight into the protective role of H2S in Cr-stressed cauliflower and suggest H2S as a potential candidate in reducing Cr toxicity in cauliflower and other crops.  相似文献   
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