scholarly journals An improved Xer-cise technology for the generation of multiple unmarked mutants in Mycobacteria

BioTechniques ◽  
2020 ◽  
Vol 68 (2) ◽  
pp. 106-110 ◽  
Author(s):  
Yves-Marie Boudehen ◽  
Maximilian Wallat ◽  
Philippe Rousseau ◽  
Olivier Neyrolles ◽  
Claude Gutierrez
Keyword(s):  
Antibiotic Resistance ◽  
Mycobacterium Tuberculosis ◽  
Central Region ◽  
High Frequency ◽  
Mycobacterium Smegmatis ◽  
Bacterial Species ◽  
Wild Type ◽  
Model Species ◽  
Bacterial Chromosomes ◽  
Zeocin Resistance

Xer-cise is a technique using antibiotic resistance cassettes flanked by dif sites allowing spontaneous and accurate excision from bacterial chromosomes with a high frequency through the action of the cellular recombinase XerCD. Here, we report a significant improvement of Xer-cise in Mycobacteria. Zeocin resistance cassettes flanked by variants of the natural Mycobacterium tuberculosis dif site were constructed and shown to be effective tools to construct multiple unmarked mutations in M. tuberculosis and in the model species Mycobacterium smegmatis. The dif site variants harbor mutations in the central region and can therefore not recombine with the wild-type or other variants, resulting in mutants of increased genetic stability. The herein described method should be generalizable to virtually any transformable bacterial species.

scholarly journals An improved Xer-cise technology for the generation of multiple unmarked mutants in Mycobacteria

2019 ◽  
Author(s):  
Yves-Marie Boudehen ◽  
Maximillian Wallat ◽  
Philippe Rousseau ◽  
Olivier Neyrolles ◽  
Claude Gutierrez
Keyword(s):  
Antibiotic Resistance ◽  
High Frequency ◽  
Mycobacterium Smegmatis ◽  
Bacterial Species ◽  
Wild Type ◽  
Model Species ◽  
Mycobacterial Genome ◽  
Bacterial Chromosomes ◽  
Genetic Constructs ◽  
Zeocin Resistance

SummaryXer-cise is a technique using antibiotic resistance cassettes flanked by dif sites allowing spontaneous and accurate excision from bacterial chromosomes with a high frequency through the action of the cellular recombinase XerCD. Here, we report a significant improvement of Xer-cise in Mycobacteria. Zeocin-resistance cassettes flanked by variants of the natural Mycobacterium tuberculosis dif site were constructed and shown to be effective tools to construct multiple unmarked mutations in M. tuberculosis and in the model species Mycobacterium smegmatis. The dif site variants harbor mutations in the central region and can therefore not recombine with the wild type or other variants, resulting in mutants of increased genetic stability. The herein described method should be generalizable to virtually any transformable bacterial species.Method summarydif-ZeoR-dif cassettes are used to replace non-essential genes in mycobacterial genome through recombineering. Spontaneous excision of the cassette is carried out under the action of the recombinase XerCD, resulting in unmarked deletions. Subsequent rounds of mutagenesis using cassettes flanked by a range of dif site variants allow construction of multiple mutants in which the different dif sites cannot recombine which each other, yielding stable genetic constructs.

scholarly journals Expression of Mycobacterium smegmatisPyrazinamidase in Mycobacterium tuberculosis Confers Hypersensitivity to Pyrazinamide and Related Amides

2000 ◽  
Vol 182 (19) ◽  
pp. 5479-5485 ◽  
Author(s):  
Helena I. M. Boshoff ◽  
Valerie Mizrahi
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Intracellular Localization ◽  
Wild Type ◽  
Gene Encoding ◽  
Allelic Exchange ◽  
Mutant Strains ◽  
Established Role ◽  
Amidase Gene

ABSTRACT A pyrazinamidase (PZase)-deficient pncA mutant ofMycobacterium tuberculosis, constructed by allelic exchange, was used to investigate the effects of heterologous amidase gene expression on the susceptibility of this organism to pyrazinamide (PZA) and related amides. The mutant was highly resistant to PZA (MIC, >2,000 μg/ml), in accordance with the well-established role ofpncA in the PZA susceptibility of M. tuberculosis (A. Scorpio and Y. Zhang, Nat. Med. 2:662–667, 1996). Integration of the pzaA gene encoding the major PZase/nicotinamidase from Mycobacterium smegmatis (H. I. M. Boshoff and V. Mizrahi, J. Bacteriol. 180:5809–5814, 1998) or the M. tuberculosis pncA gene into the pncAmutant complemented its PZase/nicotinamidase defect. In bothpzaA- and pncA-complemented mutant strains, the PZase activity was detected exclusively in the cytoplasm, suggesting an intracellular localization for PzaA and PncA. ThepzaA-complemented strain was hypersensitive to PZA (MIC, ≤10 μg/ml) and nicotinamide (MIC, ≥20 μg/ml) and was also sensitive to benzamide (MIC, 20 μg/ml), unlike the wild-type andpncA-complemented mutant strains, which were highly resistant to this amide (MIC, >500 μg/ml). This finding was consistent with the observation that benzamide is hydrolyzed by PzaA but not by PncA. Overexpression of PzaA also conferred sensitivity to PZA, nicotinamide, and benzamide on M. smegmatis (MIC, 150 μg/ml in all cases) and rendered Escherichia colihypersensitive for growth at low pH.

scholarly journals Wild-Type and Non-Wild-Type Mycobacterium tuberculosis MIC Distributions for the Novel Fluoroquinolone Antofloxacin Compared with Those for Ofloxacin, Levofloxacin, and Moxifloxacin

2016 ◽  
Vol 60 (9) ◽  
pp. 5232-5237 ◽  
Author(s):  
Xia Yu ◽  
Guirong Wang ◽  
Suting Chen ◽  
Guomei Wei ◽  
Yuanyuan Shang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Critical Concentration ◽  
Bacterial Species ◽  
Drug Susceptibility Testing ◽  
World Health ◽  
Wild Type ◽  
Resistance Mutations ◽  
Content Type ◽  
Lowenstein Jensen

ABSTRACTAntofloxacin (AFX) is a novel fluoroquinolone that has been approved in China for the treatment of infections caused by a variety of bacterial species. We investigated whether it could be repurposed for the treatment of tuberculosis by studying itsin vitroactivity. We determined the wild-type and non-wild-type MIC ranges for AFX as well as ofloxacin (OFX), levofloxacin (LFX), and moxifloxacin (MFX), using the microplate alamarBlue assay, of 126 clinicalMycobacterium tuberculosisstrains from Beijing, China, of which 48 were OFX resistant on the basis of drug susceptibility testing on Löwenstein-Jensen medium. The MIC distributions were correlated with mutations in the quinolone resistance-determining regions ofgyrA(Rv0006) andgyrB(Rv0005). Pharmacokinetic/pharmacodynamic (PK/PD) data for AFX were retrieved from the literature. AFX showed lower MIC levels than OFX but higher MIC levels than LFX and MFX on the basis of the tentative epidemiological cutoff values (ECOFFs) determined in this study. All strains with non-wild-type MICs for AFX harbored known resistance mutations that also resulted in non-wild-type MICs for LFX and MFX. Moreover, our data suggested that the current critical concentration of OFX for Löwenstein-Jensen medium that was recently revised by the World Health Organization might be too high, resulting in the misclassification of phenotypically non-wild-type strains with known resistance mutations as wild type. On the basis of our exploratory PK/PD calculations, the current dose of AFX is unlikely to be optimal for the treatment of tuberculosis, but higher doses could be effective.

scholarly journals Evaluation of Mycobacterium tuberculosisGenes Involved in Resistance to Killing by Human Macrophages

2000 ◽  
Vol 68 (1) ◽  
pp. 387-390 ◽  
Author(s):  
Barbara H. Miller ◽  
Thomas M. Shinnick
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Temperature ◽  
Glutamine Synthetase ◽  
Phospholipase C ◽  
Mycobacterium Smegmatis ◽  
Survival Rates ◽  
Open Reading Frame ◽  
Wild Type ◽  
Reading Frame ◽  
Human Macrophages

ABSTRACT A coinfection assay was developed to examine Mycobacterium tuberculosis genes suspected to be involved in resistance to killing by human macrophages. THP-1 macrophages were infected with a mixture of equal numbers of recombinant Mycobacterium smegmatis LR222 bacteria expressing an M. tuberculosis gene and wild-type M. smegmatis LR222 bacteria expressing the xylE gene. At various times after infection, the infected macrophages were lysed and the bacteria were plated. The resulting colonies were sprayed with catechol to determine the number of recombinant colonies and the number ofxylE-expressing colonies. M. smegmatis bacteria expressing the M. tuberculosis glutamine synthetase A (glnA) gene or open reading frame Rv2962c orRv2958c demonstrated significantly increased survival rates in THP-1 macrophages relative to those of xylE-expressing bacteria. M. smegmatis bacteria expressing M. tuberculosis genes for phospholipase C (plcA andplcB) or for high temperature requirement A (htrA) did not.

scholarly journals Protection of Mycobacterium tuberculosis from Reactive Oxygen Species Conferred by the mel2 Locus Impacts Persistence and Dissemination

2009 ◽  
Vol 77 (6) ◽  
pp. 2557-2567 ◽  
Author(s):  
Suat L. G. Cirillo ◽  
Selvakumar Subbian ◽  
Bing Chen ◽  
Torin R. Weisbrod ◽  
William R. Jacobs ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Mycobacterium Tuberculosis ◽  
Bacterial Species ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Wild Type ◽  
Primary Mechanism ◽  
Insight Into ◽  
Increased Susceptibility

ABSTRACT Persistence of Mycobacterium tuberculosis in humans represents a major roadblock to elimination of tuberculosis. We describe identification of a locus in M. tuberculosis, mel2, that displays similarity to bacterial bioluminescent loci and plays an important role during persistence in mice. We constructed a deletion of the mel2 locus and found that the mutant displays increased susceptibility to reactive oxygen species (ROS). Upon infection of mice by aerosol the mutant grows normally until the persistent stage, where it does not persist as well as wild type. Histopathological analyses show that infection with the mel2 mutant results in reduced pathology and both CFU and histopathology indicate that dissemination of the mel2 mutant to the spleen is delayed. These data along with growth in activated macrophages and infection of Phox−/− and iNOS−/− mice and bone marrow-derived macrophages suggest that the primary mechanism by which mel2 affects pathogenesis is through its ability to confer resistance to ROS. These studies provide the first insight into the mechanism of action for this novel class of genes that are related to bioluminescence genes. The role of mel2 in resistance to ROS is important for persistence and dissemination of M. tuberculosis and suggests that homologues in other bacterial species are likely to play a role in pathogenesis.

scholarly journals Identification of Mycobacterial Genes Involved in Antibiotic Sensitivity: Implications for the Treatment of Tuberculosis with β-Lactam-Containing Regimens

2017 ◽  
Vol 61 (7) ◽  
Author(s):  
Gopinath Viswanathan ◽  
Sangya Yadav ◽  
Tirumalai R. Raghunand
Keyword(s):  
Antibiotic Resistance ◽  
Mycobacterium Tuberculosis ◽  
Bactericidal Activity ◽  
Mycobacterium Smegmatis ◽  
Antibiotic Sensitivity ◽  
Mutant Library ◽  
Content Type ◽  
Factors Associated ◽  
Library Screen ◽  
Transposon Mutants

ABSTRACT In a Mycobacterium smegmatis mutant library screen, transposon mutants with insertions in fhaA, dprE2, rpsT, and parA displayed hypersusceptibility to antibiotics, including the β-lactams meropenem, ampicillin, amoxicillin, and cefotaxime. Sub-MIC levels of octoclothepin, a psychotic drug inhibiting ParA, phenocopied the parA insertion and enhanced the bactericidal activity of meropenem against Mycobacterium tuberculosis in combination with clavulanate. Our study identifies novel factors associated with antibiotic resistance, with implications in repurposing β-lactams for tuberculosis treatment.

scholarly journals Selective translation by alternative bacterial ribosomes

2020 ◽  
Vol 117 (32) ◽  
pp. 19487-19496
Author(s):  
Yu-Xiang Chen ◽  
Zhi-yu Xu ◽  
Xueliang Ge ◽  
Jia-Yao Hong ◽  
Suparna Sanyal ◽  
...  
Keyword(s):  
Mycobacterium Smegmatis ◽  
Iron Homeostasis ◽  
Bacterial Species ◽  
Ribosome Profiling ◽  
Wild Type ◽  
Initiation Complex ◽  
Selective Translation ◽  
Differential Efficiency ◽  
Hostile Environments ◽  
Type Strains

Alternative ribosome subunit proteins are prevalent in the genomes of diverse bacterial species, but their functional significance is controversial. Attempts to study microbial ribosomal heterogeneity have mostly relied on comparing wild-type strains with mutants in which subunits have been deleted, but this approach does not allow direct comparison of alternate ribosome isoforms isolated from identical cellular contexts. Here, by simultaneously purifying canonical and alternative RpsR ribosomes fromMycobacterium smegmatis, we show that alternative ribosomes have distinct translational features compared with their canonical counterparts. Both alternative and canonical ribosomes actively take part in protein synthesis, although they translate a subset of genes with differential efficiency as measured by ribosome profiling. We also show that alternative ribosomes have a relative defect in initiation complex formation. Furthermore, a strain ofM. smegmatisin which the alternative ribosome protein operon is deleted grows poorly in iron-depleted medium, uncovering a role for alternative ribosomes in iron homeostasis. Our work confirms the distinct and nonredundant contribution of alternative bacterial ribosomes for adaptation to hostile environments.

scholarly journals Ribosomal mutations promote the evolution of antibiotic resistance in a multidrug environment

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
James E Gomez ◽  
Benjamin B Kaufmann-Malaga ◽  
Carl N Wivagg ◽  
Peter B Kim ◽  
Melanie R Silvis ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Mycobacterium Smegmatis ◽  
Large Scale ◽  
Wild Type ◽  
Membrane Stress ◽  
Stepping Stones ◽  
Suppressor Mutations ◽  
Genes Encoding ◽  
High Level ◽  
Chromosomal Mutations

Antibiotic resistance arising via chromosomal mutations is typically specific to a particular antibiotic or class of antibiotics. We have identified mutations in genes encoding ribosomal components in Mycobacterium smegmatis that confer resistance to several structurally and mechanistically unrelated classes of antibiotics and enhance survival following heat shock and membrane stress. These mutations affect ribosome assembly and cause large-scale transcriptomic and proteomic changes, including the downregulation of the catalase KatG, an activating enzyme required for isoniazid sensitivity, and upregulation of WhiB7, a transcription factor involved in innate antibiotic resistance. Importantly, while these ribosomal mutations have a fitness cost in antibiotic-free medium, in a multidrug environment they promote the evolution of high-level, target-based resistance. Further, suppressor mutations can then be easily acquired to restore wild-type growth. Thus, ribosomal mutations can serve as stepping-stones in an evolutionary path leading to the emergence of high-level, multidrug resistance.

scholarly journals The RipA and RipB Peptidoglycan Endopeptidases Are Individually Nonessential to Mycobacterium smegmatis

2016 ◽  
Vol 198 (9) ◽  
pp. 1464-1475 ◽  
Author(s):  
Daniel J. Martinelli ◽  
Martin S. Pavelka
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cell Division ◽  
Mycobacterium Smegmatis ◽  
Wild Type ◽  
Content Type ◽  
Deletion Mutations ◽  
Levels Of Detail ◽  
Mutant Strains ◽  
Single Deletion ◽  
Increased Susceptibility

ABSTRACTMycobacteria possess a series of Rip peptidoglycan endopeptidases that have been characterized in various levels of detail. The RipA and RipB proteins have been extensively studied and aredl-endopeptidases, and RipA has been considered essential toMycobacterium smegmatisandMycobacterium tuberculosis. We show here that theripAandripBgenes are individually dispensable inM. smegmatisand that at least one of the genes must be expressed for viability. We characterized strains carrying in-frame deletion mutations ofripAandripBand found that both mutant strains exhibited increased susceptibility to a limited number of antibiotics and to detergent but that only the ΔripAmutant displayed hypersusceptibility to lysozyme. We also constructed and characterized ΔripDand ΔripAΔripDmutants and found that the single mutant had only an intermediate lysozyme hypersusceptibility phenotype compared to that of wild-type cells while loss ofripDin the ΔripAbackground partially rescued the antibiotic and lysozyme phenotypes of the ΔripAmutant.IMPORTANCEWe show that the RipA endopeptidase, which has been considered essential for cell division in certain mycobacteria, is not essential but that at least it or a similar protein, RipB, must be expressed by the bacteria for viability. This work is the first description of strains carrying single deletion mutations of RipA, RipB, and a novel endopeptidase-like protein, RipD.

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