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Articles by Renan Goude in JoVE
JoVE General
माइक्रोबैक्टीरिया की electroporation
1Center for Infectious Disease, Barts and the London School of Medicine and Dentistry, 2Institute for Cell and Molecular Science, Barts and the London School of Medicine and Dentistry
Mycobacterial रोगजनक रणनीति बुरा समझा रहते हैं. अधिकांश प्रजातियों की धीमी विकास दर है, सेल की दीवार के अभेद्य प्रकृति, और रोगज़नक़ों के साथ काम करने के खतरों माइक्रोबैक्टीरिया अध्ययन करने के लिए मुश्किल बनाने के लिए और हमारे इन जीवों के गरीब को समझने के लिए काफी हद तक जिम्मेदार हैं. इस वीडियो में हम electroporation की तकनीक है, जो एक संक्षिप्त उच्च विद्युत आवेग को subjecting कोशिकाओं को शामिल करने के लिए डीएनए के प्रवेश की अनुमति प्रदर्शन करेंगे. यह माइक्रोबैक्टीरियल कोशिकाओं में डीएनए को शुरू करने के लिए सबसे व्यापक रूप से इस्तेमाल किया विधि है.
Other articles by Renan Goude on PubMed
Erwinia Chrysanthemi O Antigen is Required for Betaine Osmoprotection in High-salt Media
Cellular components necessary for osmoprotection are poorly known. In this study we show that O antigen is specifically required for the effectiveness of betaines as osmoprotectants for Erwinia chrysanthemi in saline media. The phenotype is correlated with the inability of rfb mutant strains to maintain a high accumulation level of betaines in hypersaline media.
Glutamine, Glutamate, and Alpha-glucosylglycerate Are the Major Osmotic Solutes Accumulated by Erwinia Chrysanthemi Strain 3937
Erwinia chrysanthemi is a phytopathogenic soil enterobacterium closely related to Escherichia coli. Both species respond to hyperosmotic pressure and to external added osmoprotectants in a similar way. Unexpectedly, the pools of endogenous osmolytes show different compositions. Instead of the commonly accumulated glutamate and trehalose, E. chrysanthemi strain 3937 promotes the accumulation of glutamine and alpha-glucosylglycerate, which is a new osmolyte for enterobacteria, together with glutamine. The amounts of the three osmolytes increased with medium osmolarity and were reduced when betaine was provided in the growth medium. Both glutamine and glutamate showed a high rate of turnover, whereas glucosylglycerate stayed stable. In addition, the balance between the osmolytes depended on the osmolality of the medium. Glucosylglycerate and glutamate were the major intracellular compounds in low salt concentrations, whereas glutamine predominated at higher concentrations. Interestingly, the ammonium content of the medium also influenced the pool of osmolytes. During bacterial growth with 1 mM ammonium in stressing conditions, more glucosylglycerate accumulated by far than the other organic solutes. Glucosylglycerate synthesis has been described in some halophilic archaea and bacteria but not as a dominant osmolyte, and its role as an osmolyte in Erwinia chrysanthemi 3937 shows that nonhalophilic bacteria can also use ionic osmolytes.
EmbA is an Essential Arabinosyltransferase in Mycobacterium Tuberculosis
The Emb proteins (EmbA, EmbB, EmbC) are mycobacterial arabinosyltransferases involved in the biogenesis of the mycobacterial cell wall. EmbA and EmbB are predicted to work in unison as a heterodimer. EmbA and EmbB are involved in the formation of the crucial terminal hexaarabinoside motif [Arabeta(1-->2)Araalpha(1-->5)] [Arabeta(1-->2)Araalpha(1-->3)]Araalpha(1-->5)Araalpha1-->(Ara(6)) in the cell wall polysaccharide arabinogalactan. Studies conducted in Mycobacterium smegmatis revealed that mutants with disruptions in embA or embB are viable, although the growth rate was affected. In contrast, we demonstrate here that embA is an essential gene in Mycobacterium tuberculosis, since a deletion of the chromosomal gene could only be achieved when a second functional copy was provided on an integrated vector. Complementation of an embA mutant of M. smegmatis by M. tuberculosis embA confirmed that it encodes a functional arabinosyltransferase. We identified a promoter for M. tuberculosis embA located immediately upstream of the gene, indicating that it is expressed independently from the upstream gene, embC. Promoter activity from P(embA)((Mtb)) was sevenfold lower when assayed in M. smegmatis compared to M. tuberculosis, indicating that the latter is not a good host for genetic analysis of M. tuberculosis embA expression. P(embA)((Mtb)) activity remained constant throughout growth phases and after stress treatment, although it was reduced during hypoxia-induced non-replicating persistence. Ethambutol exposure had no effect on P(embA)((Mtb)) activity. These data demonstrate that M. tuberculosis embA encodes a functional arabinosyltransferase which is constitutively expressed and plays a critical role in M. tuberculosis.
The Critical Role of EmbC in Mycobacterium Tuberculosis
Arabinan polymers are major components of the cell wall in Mycobacterium tuberculosis and are involved in maintaining its structure, as well as playing a role in host-pathogen interactions. In particular, lipoarabinomannan (LAM) has multiple immunomodulatory effects. In the nonpathogenic species Mycobacterium smegmatis, EmbC has been identified as a key arabinosyltransferase involved in the incorporation of arabinose into LAM, and an embC mutant is viable but lacks LAM. In contrast, we demonstrate here that in M. tuberculosis, embC is an essential gene under normal growth conditions, suggesting a more crucial role for LAM in the pathogenic mycobacteria. M. tuberculosis EmbC has an activity similar to that of M. smegmatis EmbC, since we were able to complement an embC mutant of M. smegmatis with embC(Mtb), confirming that it encodes a functional arabinosyltransferase. In addition, we observed that the size of LAM produced in M. smegmatis was dependent on the level of expression of embC(Mtb). Northern analysis revealed that embC is expressed as part of a polycistronic message encompassing embC and three upstream genes. The promoter region for this transcript was identified and found to be up-regulated in stationary phase but down-regulated during hypoxia-induced nonreplicating persistence. In conclusion, we have identified one of the key genes involved in LAM biosynthesis in M. tuberculosis and confirmed its essential role in this species.
The Genetics of Cell Wall Biosynthesis in Mycobacterium Tuberculosis
Despite an available vaccine and effective antibiotics, Mycobacterium tuberculosis is still the causative agent of almost 2 million deaths every year. The cell wall of M. tuberculosis is composed of sugars and lipids of exotic structure, many of which contribute to its pathogenicity. The majority of the enzymes responsible for building this structure are essential. However, they share very little homology with well-characterized enzymes, which makes their identification in the genome difficult. Despite this, our knowledge of the structure of the cell wall of M. tuberculosis is fairly complete and an increasing number of genes have been identified that are involved in its biosynthesis. By contrast, data concerning regulation of the expression of these genes and control of the cell wall composition are restricted. This review summarizes current information on the genetics of cell wall biosynthesis in M. tuberculosis, incorporating available data on gene organization and regulation.
Electroporation of Mycobacteria
High-efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall but is compounded by the fact that most molecular techniques have been developed for distantly related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified, and DNA transformation of many mycobacterial species has now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the conditions for the electroporation pulse. Optimized methods for the transformation of both slow-grower and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.
RpoE2 of Sinorhizobium Meliloti is Necessary for Trehalose Synthesis and Growth in Hyperosmotic Media
Adaptation to osmotic stress can be achieved by the accumulation of compatible solutes that aid in turgor maintenance and macromolecule stabilization. The genetic regulation of solute accumulation is poorly understood, and has been described well at the molecular level only in enterobacteria. In this study, we show the importance of the alternative sigma factor RpoE2 in Sinorhizobium meliloti osmoadaptation. Construction and characterization of an S. meliloti rpoE2 mutant revealed compromised growth in hyperosmotic media. This defect was due to the lack of trehalose, a minor carbohydrate osmolyte normally produced in the initial stages of growth and in stationary phase. We demonstrate here that all three trehalose synthesis pathways are RpoE2 dependent, but only the OtsA pathway is important for osmoinducible trehalose synthesis. Furthermore, we confirm that the absence of RpoE2-dependent induction of otsA is the cause of the osmotic phenotype of the rpoE2 mutant. In conclusion, we have highlighted that, despite its low level, trehalose is a crucial compatible solute in S. meliloti, and the OtsA pathway induced by RpoE2 is needed for its accumulation under hyperosmotic conditions.
