Modeling Site-Specific Nucleotide Biases Affecting Himar1 Transposon Insertion Frequencies in TnSeq Data Sets

Choudhery, Sanjeevani; Brown, A. J. P.; Akusobi, Chidiebere; Rubín, Eric J.; Sassetti, Christopher M.; Ioerger, Thomas R.

doi:10.1128/msystems.00876-21

Cited by 10 publications

(13 citation statements)

References 30 publications

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“…Across three libraries, we generated approximately 51,000 unique transposon mutants, as defined by sites of unique transposon insertions across all three libraries. Of note, the Mab libraries shared the same non-permissive ‘TA’ sites for transposon insertion as has been described in other mycobacterial species ( Choudhery et al, 2021 ).…”

Section: Resultsmentioning

confidence: 73%

Transposon mutagenesis in Mycobacterium abscessus identifies an essential penicillin-binding protein involved in septal peptidoglycan synthesis and antibiotic sensitivity

Akusobi

Benghomari

Zhu

et al. 2022

eLife

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Mycobacterium abscessus (Mab) is a rapidly growing non-tuberculous mycobacterium (NTM) that causes a wide range of infections. Treatment of Mab infections is difficult because the bacterium is intrinsically resistant to many classes of antibiotics. Developing new and effective treatments against Mab requires a better understanding of the unique vulnerabilities that can be targeted for future drug development. To achieve this, we identified essential genes in Mab by conducting transposon sequencing (TnSeq) on the reference Mab strain ATCC 19977. We generated ~51,000 unique transposon mutants and used this high-density library to identify 362 essential genes for in vitro growth. To investigate species-specific vulnerabilities in Mab, we further characterized MAB_3167c, a predicted penicillin-binding protein and hypothetical lipoprotein (PBP-lipo) that is essential in Mab and non-essential in Mycobacterium tuberculosis (Mtb). We found that PBP-lipo primarily localizes to the subpolar region and later to the septum as cells prepare to divide. Depletion of Mab PBP-lipo causes cells to elongate, develop ectopic branches, and form multiple septa. Knockdown of PBP-lipo along with PbpB, DacB1, and a carboxypeptidase, MAB_0519 lead to synergistic growth arrest. In contrast, these genetic interactions were absent in the Mtb model organism, Mycobacterium smegmatis, indicating that the PBP-lipo homologs in the two species exist in distinct genetic networks. Finally, repressing PBP-lipo sensitized the reference strain and 11 Mab clinical isolates to several classes of antibiotics, including the β-lactams, ampicillin, and amoxicillin by greater than 128-fold. Altogether, this study presents PBP-lipo as a key enzyme to study Mab-specific processes in cell wall synthesis and importantly positions PBP-lipo as an attractive drug target to treat Mab infections.

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Section: Resultsmentioning

confidence: 73%

Transposon mutagenesis in Mycobacterium abscessus identifies an essential penicillin-binding protein involved in septal peptidoglycan synthesis and antibiotic sensitivity

Akusobi

Benghomari

Zhu

et al. 2022

eLife

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show abstract

Section: Resultsmentioning

confidence: 73%

High-density transposon mutagenesis in Mycobacterium abscessus identifies an essential penicillin-binding lipo-protein (PBP-lipo) involved in septal peptidoglycan synthesis and antibiotic sensitivity

Akusobi

et al. 2021

Preprint

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Mycobacterium abscessus (Mab) is a rapidly growing non-tuberculous mycobacterium (NTM) that causes a wide range of infections. Treatment of Mab infections is difficult because the bacterium is intrinsically resistant to many classes of antibiotics. Developing new and effective treatments against Mab requires a better understanding of the unique vulnerabilities that can be targeted for future drug development. To achieve this, we identified essential genes in Mab by conducting transposon-sequencing (TnSeq) on the reference Mab strain ATCC 19977. We generated ~51,000 unique transposon mutants and used this high-density library to identify 362 essential genes for in vitro growth. To investigate species-specific vulnerabilities in Mab, we further characterized MAB_3167c, a predicted penicillin-binding-lipoprotein (PBP-lipo) that is essential in Mab and non-essential in M. tuberculosis (Mtb). We found that PBP-lipo primarily localizes to the subpolar region and later to the septum as cells prepare to divide. Knockdown of Mab PBP-lipo causes cells to elongate, develop ectopic branches, and form multiple septa. Dual knockdown of PBP-lipo with PBPs, PbpB, DacB1, and a carboxypeptidase, MAB_0519 lead to synergistic growth arrest. In contrast, these genetic interactions were absent in the Mtb model organism, M. smegmatis, indicating that the PBP-lipo homologs in the two species exist in distinct genetic networks. Finally, repressing PBP-lipo sensitized the reference strain and 11 Mab clinical isolates to several classes of antibiotics, including the beta-lactams, ampicillin and amoxicillin by greater than 128-fold. Altogether, this study presents PBP-lipo as a key enzyme to study Mab specific processes in cell wall synthesis and importantly positions PBP-lipo as an attractive drug target to treat Mab infections.

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“…A widely used approach is the use of transposons that enable the random integration of DNA segments across the genome without the need for homology [ 90 ]. Examples of transposons used for random DNA insertion include Himar1 , which inserts randomly between any TA dinucleotide, and Tn5, which can insert in the genome mostly at random but has a bias towards certain DNA shapes and GC rich or AT rich regions [ 136 , 137 ]; therefore, Tn5 insertion is not uniformly random. The combination of random transposon mutagenesis and high-throughput sequencing for genome-scale analyses is referred to as transposon sequencing (TnSeq).…”

Section: Genetic Design Tools Used To Engineer Probioticsmentioning

confidence: 99%

Recent advances in genetic tools for engineering probiotic lactic acid bacteria

et al. 2023

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Synthetic biology has grown exponentially in the last few years, with a variety of biological applications. One of the emerging applications of synthetic biology is to exploit the link between microorganisms, biologics, and human health. To exploit this link, it is critical to select effective synthetic biology tools for use in appropriate microorganisms that would address unmet needs in human health through the development of new game-changing applications and by complementing existing technological capabilities. Lactic acid bacteria (LAB) are considered appropriate chassis organisms that can be genetically engineered for therapeutic and industrial applications. Here, we have reviewed comprehensively various synthetic biology techniques for engineering probiotic LAB strains, such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 mediated genome editing , homologous recombination, and recombineering. In addition, we also discussed heterologous protein expression systems used in engineering probiotic LAB. By combining computational biology with genetic engineering, there is a lot of potential to develop next-generation synthetic LAB with capabilities to address bottlenecks in industrial scale-up and complex biologics production. Recently, we started working on Lactochassis project where we aim to develop next generation synthetic LAB for biomedical application.

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Modeling Site-Specific Nucleotide Biases Affecting Himar1 Transposon Insertion Frequencies in TnSeq Data Sets

Cited by 10 publications

References 30 publications

Transposon mutagenesis in Mycobacterium abscessus identifies an essential penicillin-binding protein involved in septal peptidoglycan synthesis and antibiotic sensitivity

Transposon mutagenesis in Mycobacterium abscessus identifies an essential penicillin-binding protein involved in septal peptidoglycan synthesis and antibiotic sensitivity

High-density transposon mutagenesis in Mycobacterium abscessus identifies an essential penicillin-binding lipo-protein (PBP-lipo) involved in septal peptidoglycan synthesis and antibiotic sensitivity

Recent advances in genetic tools for engineering probiotic lactic acid bacteria

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