FLUTE Experiment 2

by patricwpadmin

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
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)

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

Conditional Essentials:

The following genes are indicated as conditional essentials based on statistical analysis (resampling) output using Transit software (http://saclab.tamu.edu/essentiality/transit/). 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.
resampling_dPonA1_wt data set
ORF log2 FC q-value Feature in PATRIC
Rv0007 -8.44 0 Feature page
Rv0050 -9.59 0 Feature page
Rv0096 -3.59 0 Feature page
Rv0097 -4.03 0 Feature page
Rv0101 -1.74 0 Feature page
Rv0127 -3.35 0 Feature page
Rv0155 -6.58 0 Feature page
Rv0157 -5.93 0 Feature page
Rv0211 -6.04 0 Feature page
Rv0238 -8.61 0 Feature page
Rv0455c -5.73 0 Feature page
Rv0467 -6.84 0 Feature page
Rv0489 -5.36 0 Feature page
Rv0642c -4.97 0 Feature page
Rv0643c -2.38 0 Feature page
Rv0806c -7.42 0 Feature page
Rv0860 -2.83 0 Feature page
Rv1086 -7.96 0 Feature page
Rv1112 -4.18 0 Feature page
Rv1339 -4.55 0 Feature page
Rv1421 -2.29 0 Feature page
Rv1565c -5.85 0 Feature page
Rv1798 -3.72 0 Feature page
Rv1836c -2.57 0 Feature page
Rv2140c -5.52 0 Feature page
Rv2171 -8.78 0 Feature page
Rv2176 -3.78 0 Feature page
Rv2222c -1.95 0 Feature page
Rv2224c -3.49 0 Feature page
Rv2404c -5.02 0 Feature page
Rv2535c -4.47 0 Feature page
Rv2864c -2.86 0 Feature page
Rv3302c 10.81 0 Feature page
Rv3484 -1.96 0 Feature page
Rv3490 -4.67 0 Feature page
Rv3682 -8.89 0 Feature page
Rv3910 -4.88 0 Feature page
Rv0066c -3.32 0.0095 Feature page
Rv0153c -4.02 0.0095 Feature page
Rv1410c -2.08 0.0095 Feature page
Rv1432 -3.7 0.0095 Feature page
Rv1780 -1.98 0.0095 Feature page
Rv1248c -3.79 0.017 Feature page
Rv1371 -3.3 0.017 Feature page
Rv2038c -3.26 0.017 Feature page
Rv2940c -1.08 0.017 Feature page
Rv3529c -2.95 0.017 Feature page
Rv1662 -2.85 0.0249 Feature page
Rv0180c 8.32 0.0307 Feature page
Rv1183 -1.54 0.0307 Feature page
Rv2246 -4.65 0.0307 Feature page
Rv3210c -4.47 0.0307 Feature page
Rv1401 -2.81 0.0369 Feature page
Rv2462c -1.79 0.0369 Feature page
Rv0260c -2.99 0.0413 Feature page
Rv1220c -3.27 0.0413 Feature page
Rv1791 -7.46 0.0413 Feature page
Rv2809 -3.36 0.0413 Feature page
Rv2131c -3.93 0.0473 Feature page