FLUTE Experiment 2

Published on 2016-01-21

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

Publication: PMCID: PMC4620856

Peptidoglycan (PG), a complex polymer composed of saccharide chains crosslinked by short peptides, is a critical component of the bacterial cell wall. PG synthesis has been extensively studied in model organisms but remains poorly understood in mycobacteria, a genus that includes the important human pathogen Mycobacterium tuberculosis (Mtb). The principle PG synthetic enzymes have similar and, at times, overlapping functions. To determine how these are functionally organized,we carried out whole genome transposon mutagenesis screens in Mtb strains deleted for ponA1, ponA2, and ldtB, major PG synthetic enzymes. We identified distinct factors required to sustain bacterial growth in the absence of each of these enzymes. We find that even the homologues PonA1 and PonA2 have unique sets of genetic interactions, suggesting there are distinct PG synthesis pathways in Mtb. Either PonA1 or PonA2 is required for growth of Mtb, but both genetically interact with LdtB, which has its own distinct genetic network. We further provide evidence that each interaction network is differentially susceptible to antibiotics. Thus, Mtb uses alternative pathways to produce PG, each with its own biochemical characteristics and vulnerabilities.

Experimenter/researcher/owner of data: Karen Kieser PI/lab: Eric Rubin, Harvard School of Public Health Uploaded/deposited by: Thomas Ioerger, Texas A&M University Publication: manuscript in preparation (K. Kieser et al.) Dataset: dPonA1_KOD.wig SRA accession: SRA277968 http://www.ncbi.nlm.nih.gov/sra/?term=SRA277968[accn] Library: This TnSeq library consists of insertion mutants of the Himar1 transposon in a Rv0050/PonA1-deletion mutant of the M. tuberculosis H37Rv strain, constructed by Karen Kieser in the Rubin lab. Conditions: grown on plates with 7H10 medium Transposon used: Himar1 Protocol for library preparation: Himar1 transfection as described in Long et al (2015). Protocol for TnSeq sample preparation: nested PCR, as described in Long et al (2015). Protocol for DNA extraction: (perhaps not relevant) Protocol for sequencing: 54 bp paired-end reads on Illumina GAII (sequencing date: 4/2/2013) Protocol for data processing: mapped reads to reference genome using TRANSIT (Ioerger et al., 2015); Reported as unique template counts at TA dinucleotides Reference genome: M. tuberculosis H37Rv (GenBank refseq accession number: NC_000962.2)

References:

Long, J.E., DeJesus, M., Ward, D., Baker, R.E., Ioerger, T.R. and Sassetti, C.M. (2015). Identifying essential genes in Mycobacterium

tuberculosis by global phenotypic profiling. in: Methods in Molecular Biology: Gene Essentiality, (Long Jason Lu, ed.), vol. 1279.

DeJesus, M.A., Ambadipudi, C., Baker, R., Sassetti, C., and Ioerger, T.R. (2015). TRANSIT – a Software Tool for Himar1 TnSeq Analysis. PLOS Computational Biology, to appear.

Results Dataset:  `resampling_dPonA1_wt.xlsx <http://brcdownloads.patricbrc.org/BRC_Mirrors/FLUTE/resampling_dPonA1_wt.xlsx>`__

Conditional Essentials:

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      The following genes are indicated as conditional essentials based on statistical analysis (resampling) output using Transit software (<a href="http://saclab.tamu.edu/essentiality/transit/">http://saclab.tamu.edu/essentiality/transit/</a>). In this method, for each ORF (e.g., Rv0001) Transit calculates to determine whether the essentiality of the gene significantly increase or decreases. The adjusted p-value uses the Benjamini-Hochberg correction for multiple tests, with a threshold of <0.05 for significance.
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      <strong>resampling_dPonA1_wt data set</strong>
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ORF

log2 FC

q-value

Feature in PATRIC

Rv0007

-8.44

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Rv0050

-9.59

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Rv0096

-3.59

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Rv0097

-4.03

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Rv0101

-1.74

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Rv0127

-3.35

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Rv0155

-6.58

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Rv0157

-5.93

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Rv0211

-6.04

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Rv0238

-8.61

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Rv0455c

-5.73

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Rv0467

-6.84

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Rv0489

-5.36

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Rv0642c

-4.97

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Rv0643c

-2.38

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Rv0806c

-7.42

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Rv0860

-2.83

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Rv1086

-7.96

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Rv1112

-4.18

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Rv1339

-4.55

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Rv1421

-2.29

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Rv1565c

-5.85

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Rv1798

-3.72

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Rv1836c

-2.57

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Rv2140c

-5.52

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Rv2171

-8.78

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Rv2176

-3.78

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Rv2222c

-1.95

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Rv2224c

-3.49

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Rv2404c

-5.02

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Rv2535c

-4.47

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Rv2864c

-2.86

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Rv3302c

10.81

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Rv3484

-1.96

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Rv3490

-4.67

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Rv3682

-8.89

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Rv3910

-4.88

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Rv0066c

-3.32

0.0095

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Rv0153c

-4.02

0.0095

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Rv1410c

-2.08

0.0095

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Rv1432

-3.7

0.0095

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Rv1780

-1.98

0.0095

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Rv1248c

-3.79

0.017

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Rv1371

-3.3

0.017

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Rv2038c

-3.26

0.017

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Rv2940c

-1.08

0.017

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Rv3529c

-2.95

0.017

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Rv1662

-2.85

0.0249

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Rv0180c

8.32

0.0307

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Rv1183

-1.54

0.0307

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Rv2246

-4.65

0.0307

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Rv3210c

-4.47

0.0307

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Rv1401

-2.81

0.0369

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Rv2462c

-1.79

0.0369

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Rv0260c

-2.99

0.0413

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Rv1220c

-3.27

0.0413

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Rv1791

-7.46

0.0413

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Rv2809

-3.36

0.0413

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Rv2131c

-3.93

0.0473

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