Peptidoglycan synthesis in
            <i>Mycobacterium tuberculosis</i>
            is organized into networks with varying drug susceptibility

Kieser, Karen J.; Baranowski, Catherine; Chao, Michael C.; Long, Jarukit E.; Sassetti, Christopher M.; Waldor, Matthew K.; Sacchettini, James C.; Ioerger, Thomas R.; Rubín, Eric J.

doi:10.1073/pnas.1514135112

Cited by 82 publications

(73 citation statements)

References 34 publications

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“…Genomic DNA (gDNA) was harvested from the transposon libraries and prepared for PCR amplification as described in 20 . PCR-amplified transposon–gDNA junctions were then subjected to high-throughput sequencing using the MiSeq platform per manufacturers instructions.…”

Section: Methodsmentioning

confidence: 99%

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Deletion of a mycobacterial divisome factor collapses single-cell phenotypic heterogeneity

Rego

Audette

Rubín

2017

Nature

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158

177

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Summary Paragraph While microorganisms are often studied as populations, the behavior of single, individual cells can have profound consequences. For example, tuberculosis, caused by the bacterial pathogen Mycobacterium tuberculosis, requires months of antibiotic therapy even though the bulk of the bacterial population rapidly dies. Shorter courses lead to high rates of relapse because subpopulations of bacilli can survive despite being genetically identical to those that are easily killed 1. In fact, mycobacteria create variability every time a cell divides, producing daughter cells with different sizes and growth rates 2, 3. The mechanism(s) that underlie this high-frequency variation and how variability relates to survival of the population are unknown. Here we show that mycobacteria actively create heterogeneity. Using a fluorescent reporter and a FACS-based transposon screen, we find that deletion of lamA, a gene of previously unknown function, decreases the amount of heterogeneity in the population by decreasing asymmetric polar growth. LamA has no known homologs in other organisms, but is highly conserved across mycobacterial species. We find that LamA is a member of the mycobacterial division complex (“the divisome”). It inhibits growth at nascent new poles, creating asymmetry in polar growth. The kinetics of killing individual cells that lack lamA are more uniform and more rapid with rifampicin and certain drugs that target the cell wall. Our results show that mycobacteria encode a non-conserved protein that controls the pattern of cell growth, resulting in a population that is both heterogeneous and better able to survive antibiotic pressure.

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Section: Methodsmentioning

confidence: 99%

“…Genomic DNA-transposon junctions were mapped on the M.smegmatis genome and counted as described in 20 . The reads at each TA site for each bin where then imported into Matlab where they were processed by a custom Matlab program.…”

Section: Methodsmentioning

confidence: 99%

Deletion of a mycobacterial divisome factor collapses single-cell phenotypic heterogeneity

Rego

Audette

Rubín

2017

Nature

Self Cite

158

177

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“…As a consequence, biosynthesis of mycolic acids, the most abundant lipid of the mycobacterial outer membrane, is inhibited, causing an aberrant and lethal deformation of the cell wall (6). Recent research efforts have emphasized that also other essential steps in the biogenesis of the mycobacterial cell wall can serve as new targets for anti-tuberculosis interventions (7,8).…”

Section: And Isoniazid (Inh)mentioning

confidence: 99%

iniBAC induction Is Vitamin B12- and MutAB-dependent in Mycobacterium marinum

Boot

Sparrius

Jim

et al. 2016

Journal of Biological Chemistry

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Tuberculosis can be treated with a 6-month regimen of antibiotics. Although the targets of most of the first-line antibiotics have been identified, less research has focused on the intrabacterial stress responses that follow upon treatment with antibiotics. Studying the roles of these stress genes may lead to the identification of crucial stress-coping mechanisms that can provide additional drug targets to increase treatment efficacy. A threegene operon with unknown function that is strongly up-regulated upon treatment with isoniazid and ethambutol is the ini-BAC operon. We have reproduced these findings and show that iniBAC genes are also induced in infected host cells, although with higher variability. Next, we set out to elucidate the genetic network that results in iniBAC induction in Mycobacterium marinum. By transposon mutagenesis, we identified that the operon is highly induced by mutations in genes encoding enzymes of the vitamin B12 biosynthesis pathway and the vitamin B12-dependent methylmalonyl-CoA-mutase MutAB. Lipid analysis showed that a mutA::tn mutant has decreased phthiocerol dimycocerosates levels, suggesting a link between iniBAC induction and the production of methyl-branched lipids. Moreover, a similar screen in Mycobacterium bovis BCG identified that phthiocerol dimycocerosate biosynthesis mutants cause the up-regulation of iniBAC genes. Based on these data, we propose that iniBAC is induced in response to mutations that cause defects in the biosynthesis of methyl-branched lipids. The resulting metabolic stress caused by these mutations or caused by ethambutol or isoniazid treatment may be relieved by iniBAC to increase the chance of bacterial survival.

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“…Comparative analysis of genes that are essential under different environmental conditions has been used to dissect specific metabolic pathways (4) and mechanisms of pathogenesis (5, 6). Similarly, comparing the effects of Tn insertion in different genetic backgrounds can reveal genetic interactions that imply functional relationships between genes (7–9). …”

Section: Introductionmentioning

confidence: 99%

Comprehensive Essentiality Analysis of the Mycobacterium tuberculosis Genome via Saturating Transposon Mutagenesis

DeJesus

Gerrick

et al. 2017

mBio

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502

661

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For decades, identifying the regions of a bacterial chromosome that are necessary for viability has relied on mapping integration sites in libraries of random transposon mutants to find loci that are unable to sustain insertion. To date, these studies have analyzed subsaturated libraries, necessitating the application of statistical methods to estimate the likelihood that a gap in transposon coverage is the result of biological selection and not the stochasticity of insertion. As a result, the essentiality of many genomic features, particularly small ones, could not be reliably assessed. We sought to overcome this limitation by creating a completely saturated transposon library in Mycobacterium tuberculosis. In assessing the composition of this highly saturated library by deep sequencing, we discovered that a previously unknown sequence bias of the Himar1 element rendered approximately 9% of potential TA dinucleotide insertion sites less permissible for insertion. We used a hidden Markov model of essentiality that accounted for this unanticipated bias, allowing us to confidently evaluate the essentiality of features that contained as few as 2 TA sites, including open reading frames (ORF), experimentally identified noncoding RNAs, methylation sites, and promoters. In addition, several essential regions that did not correspond to known features were identified, suggesting uncharacterized functions that are necessary for growth. This work provides an authoritative catalog of essential regions of the M. tuberculosis genome and a statistical framework for applying saturating mutagenesis to other bacteria.

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Peptidoglycan synthesis in Mycobacterium tuberculosis is organized into networks with varying drug susceptibility

Cited by 82 publications

References 34 publications

Deletion of a mycobacterial divisome factor collapses single-cell phenotypic heterogeneity

Deletion of a mycobacterial divisome factor collapses single-cell phenotypic heterogeneity

iniBAC induction Is Vitamin B12- and MutAB-dependent in Mycobacterium marinum

Comprehensive Essentiality Analysis of the Mycobacterium tuberculosis Genome via Saturating Transposon Mutagenesis

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