Directed mutagenesis of Mycobacterium smegmatis 16S rRNA to reconstruct the in vivo evolution of aminoglycoside resistance in Mycobacterium tuberculosis

ABSTRACT Mycobacterium tuberculosis protein pairs Rv1246c-Rv1247c, Rv2865-Rv2866, and Rv3357-Rv3358, here named RelBE, RelFG, and RelJK, respectively, were identified based on homology to the Escherichia coli RelBE toxin:antitoxin (TA) module. In this study, we have characterized each Rel protein pair and have established that they are functional TA modules. Overexpression of individual M. tuberculosis rel toxin genes relE, relG, and relK induced growth arrest in Mycobacterium smegmatis; a phenotype that was completely reversible by expression of their cognate antitoxin genes, relB, relF, and relJ, respectively. We also provide evidence that RelB and RelE interact directly, both in vitro and in vivo. Analysis of the genetic organization and regulation established that relBE, relFG, and relJK form bicistronic operons that are cotranscribed and autoregulated, in a manner unlike typical TA modules. RelB and RelF act as transcriptional activators, inducing expression of their respective promoters. However, RelBE, RelFG, and RelJK (together) repress expression to basal levels of activity, while RelJ represses promoter activity altogether. Finally, we have determined that all six rel genes are expressed in broth-grown M. tuberculosis, whereas relE, relF, and relK are expressed during infection of human macrophages. This is the first demonstration of M. tuberculosis expressing TA modules in broth culture and during infection of human macrophages.

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Identification of novel diphenyl urea inhibitors of Mt-GuaB2 active against Mycobacterium tuberculosis

Microbiology ◽

10.1099/mic.0.042549-0 ◽

2011 ◽

Vol 157 (1) ◽

pp. 290-299 ◽

Cited By ~ 26

Author(s):

Veeraraghavan Usha ◽

Sudagar S. Gurcha ◽

Andrew L. Lovering ◽

Adrian J. Lloyd ◽

Athina Papaemmanouil ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Mycobacterium Smegmatis ◽

Genomic Sequence ◽

Fold Increase ◽

Inosine Monophosphate Dehydrogenase ◽

Inosine Monophosphate ◽

Genes Encoding ◽

In Trans

In contrast with most bacteria, which harbour a single inosine monophosphate dehydrogenase (IMPDH) gene, the genomic sequence of Mycobacterium tuberculosis H37Rv predicts three genes encoding IMPDH: guaB1, guaB2 and guaB3. These three genes were cloned and expressed in Escherichia coli to evaluate functional IMPDH activity. Purified recombinant Mt-GuaB2, which uses inosine monophosphate as a substrate, was identified as the only active GuaB orthologue in M. tuberculosis and showed optimal activity at pH 8.5 and 37 °C. Mt-GuaB2 was inhibited significantly in vitro by a panel of diphenyl urea-based derivatives, which were also potent anti-mycobacterial agents against M. tuberculosis and Mycobacterium smegmatis, with MICs in the range of 0.2–0.5 μg ml−1. When Mt-GuaB2 was overexpressed on a plasmid in trans in M. smegmatis, a diphenyl urea analogue showed a 16-fold increase in MIC. Interestingly, when Mt-GuaB orthologues (Mt-GuaB1 and 3) were also overexpressed on a plasmid in trans in M. smegmatis, they also conferred resistance, suggesting that although these Mt-GuaB orthologues were inactive in vitro, they presumably titrate the effect of the inhibitory properties of the active compounds in vivo.

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Characterization of Mycobacterium smegmatis Expressing the Mycobacterium tuberculosis Fatty Acid Synthase I (fas1) Gene

Journal of Bacteriology ◽

10.1128/jb.186.13.4051-4055.2004 ◽

2004 ◽

Vol 186 (13) ◽

pp. 4051-4055 ◽

Cited By ~ 47

Author(s):

Oren Zimhony ◽

Catherine Vilchèze ◽

William R. Jacobs

Keyword(s):

Mycobacterium Tuberculosis ◽

Fatty Acid ◽

Mycobacterium Smegmatis ◽

Fatty Acid Synthase ◽

Complex Interaction ◽

Fatty Acid Elongation ◽

Biochemical Studies

ABSTRACT Unlike most other bacteria, mycobacteria make fatty acids with the multidomain enzyme eukaryote-like fatty acid synthase I (FASI). Previous studies have demonstrated that the tuberculosis drug pyrazinamide and 5-chloro-pyrazinamide target FASI activity. Biochemical studies have revealed that in addition to C16:0, Mycobacterium tuberculosis FASI synthesizes C26:0 fatty acid, while the Mycobacterium smegmatis enzyme makes C24:0 fatty acid. In order to express M. tuberculosis FASI in a rapidly growing Mycobacterium and to characterize the M. tuberculosis FASI in vivo, we constructed an M. smegmatis Δfas1 strain which contained the M. tuberculosis fas1 homologue. The M. smegmatis Δfas1 (attB::M. tuberculosis fas1) strain grew more slowly than the parental M. smegmatis strain and was more susceptible to 5-chloro-pyrazinamide. Surprisingly, while the M. smegmatis Δfas1 (attB::M. tuberculosis fas1) strain produced C26:0, it predominantly produced C24:0. These results suggest that the fatty acid elongation that produces C24:0 or C26:0 in vivo is due to a complex interaction among FASI, FabH, and FASII and possibly other systems and is not solely due to FASI elongation, as previously suggested by in vitro studies.

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In Vivo Splicing and Functional Characterization ofMycobacterium leprae RecA

Journal of Bacteriology ◽

10.1128/jb.182.12.3590-3592.2000 ◽

2000 ◽

Vol 182 (12) ◽

pp. 3590-3592 ◽

Cited By ~ 7

Author(s):

Klaus Frischkorn ◽

Burkhard Springer ◽

Erik C. Böttger ◽

Elaine O. Davis ◽

M. Joseph Colston ◽

...

Keyword(s):

Escherichia Coli ◽

Mycobacterium Tuberculosis ◽

Homologous Recombination ◽

Mycobacterium Smegmatis ◽

Functional Characterization ◽

Mycobacterium Leprae ◽

Dna Damaging Agents

ABSTRACT The RecA proteins from Mycobacterium tuberculosis andMycobacterium leprae contain inteins. In contrast to theM. tuberculosis RecA, the M. leprae RecA is not spliced in Escherichia coli. We demonstrate here thatM. leprae RecA is functionally spliced inMycobacterium smegmatis and produces resistance toward DNA-damaging agents and homologous recombination.

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Use of a Tetracycline-Inducible System for Conditional Expression in Mycobacterium tuberculosis and Mycobacterium smegmatis

Applied and Environmental Microbiology ◽

10.1128/aem.71.6.3077-3084.2005 ◽

2005 ◽

Vol 71 (6) ◽

pp. 3077-3084 ◽

Cited By ~ 68

Author(s):

Paul Carroll ◽

D. G. Niranjala Muttucumaru ◽

Tanya Parish

Keyword(s):

Mycobacterium Tuberculosis ◽

Reporter Gene ◽

Mycobacterium Smegmatis ◽

Expression System ◽

Inducible Promoter ◽

Inducible Expression ◽

Essential Genes ◽

In Vivo Studies ◽

Conditional Expression

ABSTRACT A number of essential genes have been identified in mycobacteria, but methods to study these genes have not been developed, leaving us unable to determine the function or biology of the genes. We investigated the use of a tetracycline-inducible expression system in Mycobacterium tuberculosis and Mycobacterium smegmatis. Using a reporter gene which encodes an unstable variant of GFP, we showed that tetracycline-inducible expression occurred in M. smegmatis and that expression levels were titratable to some extent by varying the concentration of tetracycline. The removal of tetracycline led to cessation of GFP expression, and we showed that this was a controllable on/off switch for fluorescence upon addition and removal of the antibiotic inducer. The system also functioned in M. tuberculosis, giving inducible expression of the reporter gene. We used homologous recombination to construct a strain of M. tuberculosis that expressed the only copy of the tryptophan biosynthetic enzyme, TrpD, from the tetracycline-inducible promoter. This strain was conditionally auxotrophic, showing auxotrophy only in the absence of tetracycline, confirming that trpD was tightly controlled by the foreign promoter. This is the first demonstration of the use of an inducible promoter to generate a conditional auxotroph of M. tuberculosis. The ability to tightly regulate genes now gives us the possibility to define the functions of essential genes by switching them off under defined conditions and paves the way for in vivo studies.

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Unique C-terminal extension and interactome of Mycobacterium tuberculosis GlmU impacts its in vivo function and the survival of the pathogen

Biochemical Journal ◽

10.1042/bcj20210170 ◽

2021 ◽

Author(s):

Meetu Kumar Agarwal ◽

Vijay Kumar Soni ◽

Suresh Kumar ◽

Biplab Singha ◽

Vinay Kumar Nandicoori

Keyword(s):

Cell Wall ◽

Mycobacterium Tuberculosis ◽

Mycobacterium Smegmatis ◽

Biological Function ◽

Deletion Mutants ◽

Bifunctional Enzyme ◽

Uridine Diphosphate ◽

Carboxy Terminal ◽

Wall Components

N-acetyl glucosamine-1-phosphate uridyltransferase (GlmU) is a bifunctional enzyme involved in the biosynthesis of Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc is a critical precursor for the synthesis of peptidoglycan and other cell wall components. The absence of a homolog in eukaryotes makes GlmU an attractive target for therapeutic intervention. Mycobacterium tuberculosis GlmU (GlmUMt) has features, such as a C-terminal extension, that are not present in GlmU orthologs from other bacteria. Here, we set out to determine the uniqueness of GlmUMt by performing in vivo complementation experiments using RvDglmU mutant. We find that any deletion of the carboxy-terminal extension region of GlmUMt abolishes its ability to complement the function of GlmUMt. Results show orthologs of GlmU, including its closest ortholog, from Mycobacterium smegmatis, cannot complement the function of GlmUMt. Furthermore, the co-expression of GlmUMt domain deletion mutants with either acetyl or uridyltransferase activities failed to rescue the function. However, co-expression of GlmUMt point mutants with either acetyl or uridyltransferase activities successfully restored the biological function of GlmUMt, likely due to the formation of heterotrimers. Based on the interactome experiments, we speculate that GlmUMt participates in unique interactions essential for its in vivo function.

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Identification of the Mycobacterium tuberculosis GlnE promoter and its response to nitrogen availability

Microbiology ◽

10.1099/mic.0.28942-0 ◽

2006 ◽

Vol 152 (9) ◽

pp. 2727-2734 ◽

Cited By ~ 16

Author(s):

Carey A. Pashley ◽

Amanda C. Brown ◽

Dina Robertson ◽

Tanya Parish

Keyword(s):

Mycobacterium Tuberculosis ◽

Mycobacterium Smegmatis ◽

Nitrogen Availability ◽

Essential Gene ◽

Transcriptional Start Site ◽

Enzymic Activity ◽

Site Directed Mutagenesis ◽

Ammonia Assimilation ◽

Directed Mutagenesis ◽

Key Enzyme

Adenylyltransferase, GlnE, has a predicted role in controlling the enzymic activity of glutamine synthetase, the key enzyme in ammonia assimilation. It was previously demonstrated that glnE is an essential gene in Mycobacterium tuberculosis. glnE is located downstream of glnA2, one of four glutamine synthetases. The expression of GlnE under various conditions was determined. Although a co-transcript of glnA2 and glnE was detectable, the major transcript was monocistronic. A transcriptional start site immediately upstream of glnE was identified and it was shown by site-directed mutagenesis that the predicted −10 region is a functional promoter. It was demonstrated that in a Mycobacterium smegmatis background M. tuberculosis PglnE was up-regulated in ammonia- or glutamine-containing media.

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Deletion of RsmE 16S rRNA methyltransferase leads to low level increase in aminoglycoside resistance in Mycobacterium smegmatis

10.1101/2020.01.15.907279 ◽

2020 ◽

Cited By ~ 1

Author(s):

Shipra Bijpuria ◽

Rakesh Sharma ◽

Bhupesh Taneja

Keyword(s):

Drug Resistance ◽

16S Rrna ◽

Mycobacterium Smegmatis ◽

Drug Response ◽

Fold Increase ◽

Methylation Pattern ◽

Cross Resistance ◽

Ribosomal Protein Genes ◽

Aminoglycoside Resistance ◽

Low Level

AbstractOwing to its central role in cellular function, ribosome is one of the most common targets of antibiotics in bacteria. Mutations in rrs gene, ribosomal protein genes, methyltransfersases or drug influx/efflux are often found to overcome the drug response. Despite modulation of methylation pattern in the ribosome through mutations in the methyltransferases as one of key modulators of drug response, rsmG (gidB) is the only conserved methyltransferase associated with low-level drug resistance in large number of mycobacterial isolates. Here, we present the first evidence of association of methylation by mycobacterial RsmE, that methylates U1498 of 16S rRNA, with low levels of drug resistance. Deletion of the RsmE-homolog of Mycobacterium smegmatis leads to at least two-fold increase in the inhibitory concentration of aminoglycosides that bind in the decoding center proximal to U1498 in the 30S subunit. The change in inhibitory concentrations was highly specific and does not show any cross-resistance to drugs of other classes. Surprisingly, Rv2372c, the RsmE-homolog of Mycobacterium tuberculosis has the largest number of mutations among conserved ribosomal methyltransfersases, after gidB, highlighting the role of mutations in the RsmE methyltransferase as a key emerging mechanism of drug resistance in clinical strains of M. tuberculosis. Our work underlies the association of methylation by the RsmE-homolog with drug resistance and lays the groundwork to tackle this emerging mechanism of drug resistane in mycobacteria.

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Interaction of Erp Protein of Mycobacterium tuberculosis with Rv2212 Enhances Intracellular Survival of Mycobacterium smegmatis

Journal of Bacteriology ◽

10.1128/jb.00120-16 ◽

2016 ◽

Vol 198 (20) ◽

pp. 2841-2852 ◽

Cited By ~ 4

Author(s):

Arsheed Ahmad Ganaie ◽

Garima Trivedi ◽

Amanpreet Kaur ◽

Sidharth Shankar Jha ◽

Shashi Anand ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Adenylyl Cyclase ◽

Mycobacterium Smegmatis ◽

Second Messengers ◽

Biological Significance ◽

Intracellular Survival ◽

Interacting Protein ◽

Content Type ◽

Novel Interactions

ABSTRACTTheMycobacterium tuberculosisexportedrepetitiveprotein (RvErp) is a crucial virulence-associated factor as determined by its role in the survival and multiplication of mycobacteria in cultured macrophages andin vivo. Although attempts have been made to understand the function of Erp protein, its exact role inMycobacteriumpathogenesis is still elusive. One way to determine this is by searching for novel interactions of RvErp. Using a yeast two-hybrid assay, an adenylyl cyclase (AC), Rv2212, was found to interact with RvErp. The interaction between RvErp and Rv2212 is direct and occurs at the endogenous level. The Erp protein ofMycobacterium smegmatis(MSMEG_6405, or MsErp) interacts neither with Rv2212 nor with Ms_4279, theM. smegmatishomologue of Rv2212. Deletion mutants of Rv2212 revealed its adenylyl cyclase domain to be responsible for the interaction. RvErp enhances Rv2212-mediated cyclic AMP (cAMP) production. Also, the biological significance of the interaction between RvErp and Rv2212 was demonstrated by the enhanced survival ofM. smegmatiswithin THP-1 macrophages. Taken together, these studies address a novel mechanism by which Erp executes its function.IMPORTANCERvErp is one of the important virulence factors ofM. tuberculosis. This study describes a novel function of RvErp protein ofM. tuberculosisby identifying Rv2212 as its interacting protein. Rv2212 is an adenylyl cyclase (AC) and produces cAMP, one of the prime second messengers that regulate the intracellular survival of mycobacteria. Therefore, the significance of investigating novel interactions of RvErp is paramount in unraveling the mechanisms governing the intracellular survival of mycobacteria.

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Multi-Stress Induction of the Mycobacterium tuberculosis MbcTA Bactericidal Toxin-Antitoxin System

Toxins ◽

10.3390/toxins12050329 ◽

2020 ◽

Vol 12 (5) ◽

pp. 329

Author(s):

Kanchiyaphat Ariyachaokun ◽

Anna D. Grabowska ◽

Claude Gutierrez ◽

Olivier Neyrolles

Keyword(s):

Mycobacterium Tuberculosis ◽

Mycobacterium Smegmatis ◽

Internal Standard ◽

Physical Interaction ◽

Genetic Dissection ◽

Reporter System ◽

Fluorescent Reporter ◽

Promising Target

MbcTA is a type II toxin/antitoxin (TA) system of Mycobacterium tuberculosis. The MbcT toxin triggers mycobacterial cell death in vitro and in vivo through the phosphorolysis of the essential metabolite NAD+ and its bactericidal activity is neutralized by physical interaction with its cognate antitoxin MbcA. Therefore, the MbcTA system appears as a promising target for the development of novel therapies against tuberculosis, through the identification of compounds able to antagonize or destabilize the MbcA antitoxin. Here, the expression of the mbcAT operon and its regulation were investigated. A dual fluorescent reporter system was developed, based on an integrative mycobacterial plasmid that encodes a constitutively expressed reporter, serving as an internal standard for monitoring mycobacterial gene expression, and an additional reporter, dependent on the promoter under investigation. This system was used both in M. tuberculosis and in the fast growing model species Mycobacterium smegmatis to: (i) assess the autoregulation of mbcAT; (ii) perform a genetic dissection of the mbcA promoter/operator region; and (iii) explore the regulation of mbcAT transcription from the mbcA promoter (PmbcA) in a variety of stress conditions, including in vivo in mice and in macrophages.

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