Transformation of mycobacterial species using hygromycin resistance as selectable marker

Garbe, Thomas R.; Barathi, Jaya; Barnini, Simona; Zhang, Ying; Abou-Zeid, C.; Tang, Dan; Mukherjee, Rama; Young, Douglas B.

doi:10.1099/13500872-140-1-133

Cited by 131 publications

(117 citation statements)

References 18 publications

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“…Despite many efforts in mycobacterial genetics, only a limited number of selection markers [kanamycin (Jacobs et al, 1987), pyrF (Husson et al, 1990), hygromycin (Garbe et al, 1994), sacB (Pelicic et al, 1996), apramycin (Paget and Davies, 1996) and streptomycin/spectinomycin (Baulard et al, 1996)] have been described. Recently, the rpsL gene coding for the ribosomal protein S12 has been shown to be a useful marker for gene replacement experiments (Sander et al, 1995).…”

Section: Molecular Mechanisms Underlying Macrolide Resistancementioning

confidence: 99%

The role of ribosomal RNAs in macrolide resistance

Sander

Prammananan

Meier

et al. 1997

Molecular Microbiology

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SummaryMacrolides are bacteriostatic antibiotics which interfere with the peptidyltransfer function of the ribosome. We have investigated the molecular mechanisms underlying macrolide resistance in Mycobacterium smegmatis, an eubacterium carrying two rRNA operons. Surprisingly, drug resistance was associated not with alterations in ribosomal proteins, but with a single point mutation in the peptidyltransferase region of one of the two 23S RNA genes, i.e. A2058→G or A2059→G. This mutation resulted in a heterozygous organism with a mutated and a wild-type rRNA operon respectively. Reverse transcriptase sequencing indicated the expression of both wild-type and mutated rRNAs. The mutated operon was introduced into genetically engineered rrn ¹ strains of M. smegmatis carrying a single functional rRNA operon and into parental M. smegmatis with two chromosomal rRNA operons, using gene transfer as well as gene replacement techniques. The results obtained demonstrate the dominant nature of resistance. As exemplified in our results on macrolide resistance, a complete set of genetic tools is now available, which allows questions of dominance vs. recessivity and gene dosage effects in eubacterial ribosomal nucleic acids to be addressed experimentally in vivo.

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Section: Molecular Mechanisms Underlying Macrolide Resistancementioning

confidence: 99%

The role of ribosomal RNAs in macrolide resistance

Sander

Prammananan

Meier

et al. 1997

Molecular Microbiology

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“…Plasmids used for construction of recombinant mycobacteria were based on p16R1, a shuttle vector carrying a hygromycin resistance determinant and origins of replication suitable for maintenance in mycobacteria and in Escherichia coli (35). Constructs expressing the M. tuberculosis 19-kDa Ag, and a mutant form of the protein defective in O-linked glycosylation have been described previously (19,20).…”

Section: Recombinant Constructsmentioning

confidence: 99%

“…The same sequence was also cloned into a BamHI site within a permissive loop of the M. tuberculosis superoxide dismutase (SOD) molecule as described previously (38) Recombinant constructs were prepared in E. coli DH5␣, and the identity of each construct was confirmed by restriction profile and nucleotide sequence analysis using an ABI 310 Genetic Analyzer and ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kits (Perkin-Elmer Applied Biosystems Division). Each construct was then introduced into M. vaccae or M. smegmatis by electroporation as described previously (35).…”

Section: Recombinant Constructsmentioning

confidence: 99%

Lipoprotein Access to MHC Class I Presentation During Infection of Murine Macrophages with Live Mycobacteria

Neyrolles¹,

Gould²,

Gares³

et al. 2001

The Journal of Immunology

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Following uptake by macrophages, live mycobacteria initially reside within an immature phagosome that resists acidification and retains access to recycling endosomes. Glycolipids are exported from the mycobacterial phagosome and become available for immune recognition by CD1-restricted T cells. The aim of this study was to explore the possibility that lipoproteins might similarly escape from the phagosome and act as immune targets in cells infected with live mycobacteria. We have focused on a 19-kDa lipoprotein from Mycobacterium tuberculosis that was previously shown to be recognized by CD8+ T cells. The 19-kDa Ag was found to traffic separately from live mycobacteria within infected macrophages by a pathway that was dependent on acylation of the protein. When expressed as a recombinant protein in rapid-growing mycobacteria, the 19-kDa Ag was able to deliver peptides for recognition by MHC class I-restricted T cells by a TAP-independent mechanism. Entry into the class I pathway was rapid, dependent on acylation, and could be blocked by killing the mycobacteria by heating before infection. Although the pattern of 19-kDa trafficking was similar with different mycobacterial species, preliminary experiments suggest that class I presentation is more efficient during infection with rapid-growing mycobacteria than with the slow-growing bacillus Calmette-Guérin vaccine strain.

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“…Previously we showed that M. tuberculosis SOD was highly expressed by its own promoter in another mycobacterium, M. vaccae [11]. In the current experiment, the G152A mutant SOD was similarly transformed into M. vaccae and analysed by SDS-PAGE and SOD activity gels.…”

Section: Resultsmentioning

confidence: 67%

“…Positive clones gave a single 1.1 kb KpnI fragment containing the putative mutant M. tuberculosis SOD gene, as both the PCR product and the cloning vector contained a KpnI site. The 1.1 kb KpnI fragment was then ligated into the KpnI site of the hygromycin mycobacterial shuttle vector pl6R1 [11] and transformed into E. eoli strain XLIblue. Positive clones were similarly identified with KpnI digestion and electroporated into M. vaccae together with a vector control as described in [1].…”

Section: Mutagenesis Expression and Purification Of M Tuberculosis Sodmentioning

confidence: 99%

X‐ray structure analysis of an engineered Fe‐superoxide dismutase Gly‐Ala mutant with significantly reduced stability to denaturant

et al. 1996

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We have refined the X-ray structure of a site-directed G152A mutant of the iron-dependentosuperoxide dismutase from ~lycobaeterium tuberculosis at 2.9 A resolution. The mutation which replaces a glycine residue in a surface loop with alanine was designed to alter the conformation of this loop region which has previously been shown to play a crucial structural role in quaternary interactions within the SOD tetramer. Gly-152 was targeted as it has dihedral angles (~b = 83.1 °, V = -0.3°) close to the left-handed ~-helical conformation which is rarely adopted by other amino acids except asparagine. Gly-152 was replaced by alanine as it has similar size and polarity, yet has a very low tendency to adopt similar conformations. X-ray data collection on crystals of this mutant at 2.9 A resolution and subsequent least-squares refinement to an R-value of 0.169 clearly establish that the loop conformation is unaffected. Fluorescence studies of guanidine hydrochloride denaturation establish that the mutant is 4 kcal/mol less stable than the wild-type enzyme. Our results indicate that strict conformational constraints imposed upon a region of polypeptide, due for example to interactions with a neighbouring subunit, may force an alanine residue to adopt this sterically hindered conformation with a consequent reduction in stability of the folded conformation.A,;y words: Superoxide dismutase; ~@cobacterium tuberculosis; X-ray structure; Site-directed m utagenesis

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Transformation of mycobacterial species using hygromycin resistance as selectable marker

Cited by 131 publications

References 18 publications

The role of ribosomal RNAs in macrolide resistance

The role of ribosomal RNAs in macrolide resistance

Lipoprotein Access to MHC Class I Presentation During Infection of Murine Macrophages with Live Mycobacteria

X‐ray structure analysis of an engineered Fe‐superoxide dismutase Gly‐Ala mutant with significantly reduced stability to denaturant

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