Rv1640c (lysX)

background  +5   0  -5 -10 -15 sgRNA L2FC (+ATc/–ATc) TnSeq Gene Essentiality Predictions (1, 2) Essential Growth Defect Non-Essential Growth Advantage Unknown Operon Legend (3) Operon (1) PMID: 28096490 (2) PMID: 31388080 (3) PMID: 26536359 uvrA Rv1638A Rv1639c lysS infC rpmI rplT tsnR Rv1645c
Species: M. tuberculosis
Strain: H37Rv
PATRIC Annotation: lysyl-tRNA synthetase 2 LysX
Synonyms: lysS2, lysU, mprF
M. smegmatis homologs lysS, lysS, aspS
M. abscessus homologs MAB2319c, MAB0544, MAB2865c
Gene Start: 1848524
Gene End: 1852042
Vulnerability Index (VI): -0.2030
VI Lower Bound: -2.2540
VI Upper Bound: 2.9350
[AlphaFold] [Phyre2] [UniProt] [MycoBrowser] [Orthologs] [U19] [TnSeq DB]

Essentiality:

Call Condition Type Source Notes
Non Essential in vitro TnSeq [DeJesus et al. (2017)]
Non Essential in vitro CRISPRi [Bosch & DeJesus et al. (2021)]


DNA Sequence help
TGGGCCGATCAATCGGACTTCAGGTACGCGGATGCGCTCGTTGACGCGGGTTTCAGTGCTGATGGGGCCTCCCTCGTTGGCTGCTGTTCCAGGCGCGACCAGCTTGTTGGACCCGGCCGGAATCAGGTGGGGAACACACCCAAACCATCAGCAAAAAAAGCCCTGCACAAGCAGGGCCCAATGCCGACCGATCGCGGCCAAAGCCGCCCACGCTGCACGCAGAACAGGCATCCGAGATGCCTGTGACCGGACCGCTGAACCTTGAGAAGGTGTGAAACGATCTCGTGGCTAGCGGTGGGAGTGGGACTCCACTTAACTGTCCCTGGCCTTCGCCGGGATGGTCGCGGCGTCCAGTCTAACAGCCATGACACGTCATCTAAGACAACGGCCGACATCAGCCGGTGCCCTCAGCACACCGATGCGGGGCTACAAAGGGCTGCAACGCCGGGCATTTCCCGGCTTTTGGGAATATCCTCGCGGTCTGTGACACTCACCAAACCTCGCTCGGCCACCAGGGGAAACTCCCGGTATCACTGGGTTCCCGCGGCAGCCGGCTGGACCGTCGGCGTGATCGCGACCCTGTCACTACTGGCCAGTGTGTCGCCGTTGATTCGGTGGATCATCAAGGTTCCGCGCGAATTCATCAACGATTATCTGTTCAACTTCCCCGACACCAACTTCGCGTGGTCCTTCGTGCTGGCATTGCTGGCCGCCGCGTTAACGGCACGCAAACGCATTGCCTGGTTGGTCTTGCTGGCAAATATGGTCCTGGCCGCCGTGGTCAACGCCGCCGAGATCGCAGCGGGCGGCAACACCGCCGCCGAGAGCTTCGGGGAGAATCTCGGATTCGCCGTGCATGTTGTGGCAATCGTCGTTCTGGTGCTGGGCTATCGGGAGTTCTGGGCCAAGGTCCGCAGGGGCGCGCTGTTTCGGGCGGCTGCGGTGTGGCTTGCCGGCGCGGTGGTCGGAATCGTGGCGTCTTGGGGTCTGGTCGAACTGTTTCCGGGATCACTAGCGCCGGACGAGCGGTTGGGTTACGCGGCCAACCGGGTGGTCGGATTCGCCCTCGCCGATCCCGATCTGTTCACCGGCAGACCGCACGTCTTCCTCAACGCGATCTTCGGGTTGTTCGGCGCGTTCGCGTTGATCGGGGCTGCGATCGTCTTGTTCCTATCTCAGCGCGCTGACAATGCGCTGACCGGAGAGGACGAATCCGCTATCCGCGGGTTATTGGATTTGTATGGGAAGGACGACTCACTGGGCTACTTCGCCACCCGTCGCGACAAGTCGGTGGTATTCGCATCCAGCGGCCGCGCCTGTATCACCTACCGTGTCGAGGTCGGTGTCTGCCTAGCCAGCGGCGACCCGGTAGGCGACCACAGAGCATGGCCGCAGGCCGTCGACGCGTGGCTGCGGCTGTGCCAGACCTACGGCTGGGCACCCGGCGTGATGGGCGCCAGTTCACAAGGGGCTCAGACATATCGCGAAGCGGGCCTAACCGCGCTCGAGCTGGGCGATGAGGCCATCCTGCGGCCAGCCGATTTCAAGTTGTCCGGCCCGGAGATGCGCGGGGTGCGCCAGGCCGTGACGCGGGCCCGGCGGGCGGGGTTGACGGTGCGCATCCGGCGGCATCGCGACATTGCCGAAGACGAGATGGCGCAGACCATCACACGGGCGGATTCCTGGCGCGACACCGAGACCGAGCGCGGCTTCTCGATGGCGCTGGGCCGCCTCGGCGACCCCGCGGACTCCGACTGCCTGCTGGTGGAGGCGATTGATCCGCACAACCAGGTGCTGGCAATGCTGTCGCTGGTGCCGTGGGGAACCACCGGTGTCTCCCTGGATTTGATGCGTCGTTCTCCACAATCCCCGAACGGCACTATCGAACTTATGGTCAGCGAACTCGCCTTGCACGCTGAAAGCCTTGGTATCACTCGTATTTCGTTGAACTTCGCGGTGTTTCGCGCGGCCTTCGAGCAGGGCGCCCAACTCGGCGCCGGCCCGGTTGCCCGGTTGTGGCGGGGGCTGCTGGTCTTCTTCTCACGGTGGTGGCAGCTCGAGACGCTCTACCGCTCCAACATGAAGTACCAACCCGAATGGGTACCGCGCTACGCCTGCTACGAAGACGCCCGAGTGATTCCCCGAGTGGGCGTCGCCTCGGTGATCGCAGAGGGGTTCCTCGTGCTGCCGTTCAGCCGGCGCAACCGAGTACACACGGGGCATCATCCGGCAGTGCCCGAGCGGTTGGCGGCCACCGGCCTATTGCATCACGACGGCTCGGCACCGGACGTGAGCGGGCTGCGGCAAGTAGGCCTCACCAATGGCGACGGGGTCGAGCGGCGGCTACCCGAGCAGGTGCGAGTTCGGTTCGACAAGCTAGAGAAGCTGCGGAGCAGCGGCATTGACGCCTTTCCCGTGGGCCGGCCGCCCAGTCACACGGTGGCACAGGCGCTGGCCGCAGACCACCAGGCTAGCGTGTCGGTGTCCGGACGCATCATGCGAATACGTAACTACGGTGGTGTGTTGTTCGCCCAGCTGCGCGACTGGTCGGGCGAAATGCAAGTGCTGCTGGATAATTCACGCCTGGACCAGGGCTGCGCGGCCGACTTCAACGCGGCGACCGATCTGGGTGATCTGGTGGAGATGACCGGACACATGGGCGCCAGCAAGACCGGAACTCCATCGTTGATCGTCAGCGGCTGGCGCCTGATCGGTAAGTGTTTGCGACCGTTGCCGAATAAGTGGAAGGGGCTACTGGATCCCGAAGCCCGAGTCCGGACCCGTTACCTCGACCTGGCGGTCAACGCTGAGTCCCGTGCGCTGATCACCGCTCGCAGCAGCGTGCTGCGCGCTGTGCGCGAGACGCTGTTCGCAAAGGGATTCGTCGAGGTCGAAACGCCGATCCTACAGCAGCTGCACGGCGGCGCCACCGCCCGACCGTTCGTCACGCACATCAACACGTATTCCATGGATCTGTTCCTGCGCATCGCACCAGAGCTCTACCTCAAGCGCCTGTGCGTCGGCGGCGTGGAACGGGTATTCGAGCTCGGCCGTGCCTTTCGCAACGAGGGCGTCGACTTCAGCCACAACCCGGAGTTCACCCTACTGGAGGCCTATCAAGCACACGCCGACTACCTTGAGTGGATTGACGGCTGCCGCGAACTCATCCAGAACGCCGCCCAGGCCGCCAACGGGGCGCCCATCGCCATGCGGCCCCGGACGGACAAGGGTTCCGACGGCACCCGCCACCACCTCGAACCGGTCGACATCTCCGGGATATGGCCGGTGCGGACGGTGCATGATGCGATCTCGGAAGCACTCGGTGAACGCATCGACGCCGATACCGGCTTGACCACTCTCCGCAAGCTGTGCGACGCCGCCGGTGTCCCCTATCGGACCCAATGGGATGCCGGCGCGGTCGTGCTCGAGCTATACGAGCACCTGGTGGAGTGCCGCACCGAGCAACCGACGTTCTACATCGACTTTCCGACATCGGTGTCACCGTTGACCCGACCGCATCGCAGCAAGCGCGGTGTCGCCGAGCGTTGGGACCTGGTGGCGTGGGGTATCGAGCTGGGCACCGCCTACAGCGAGCTGACCGACCCGGTGGAGCAACGGCGCCGCCTGCAGGAGCAGTCCCTGCTGGCCGCCGGCGGAGACCCCGAGGCGATGGAACTCGACGAAGACTTCCTGCAAGCGATGGAGTACGCCATGCCGCCCACCGGCGGCTTGGGCATGGGCATTGACCGGGTGGTCATGCTGATCACTGGGCGCAGCATTCGCGAAACACTGCCCTTTCCGTTGGCAAAACCGCATTAG
Vulnerability Assesment help
[Full list of passaging experiments]

Experiment: We designed an M. tuberculosis CRISPRi library (RLC12) to target all annotated Mtb genes with single guide RNAs (sgRNAs) of varying predicted knockdown efficiencies. In parallel, we constructed a M. smegmatis CRISPRi library (RLC11) to target all annotated Msmeg genes, similar to the Mtb sgRNA library. RLC12 was transformed into M. tuberculosis H37Rv; RLC11 into M. smegmatis mc2-155. We passaged both CRISPRi libraries for nearly 30 generations in the presence or absence of the CRISPRi-inducer anhydrotetracycline (ATc). At the indicated timepoints, genomic DNA was harvested and the representation of individual sgRNAs was counted using Illumina sequencing. Here, we show sgRNA log2 fold-change values (L2FC) (plus versus minus ATc) over consecutive generations for sgRNAs targeting the gene you selected. sgRNAs are color-coded based on their predicted strength from blue (strength=0; weak) to red (strength=1; strong).

In order to quantify gene vulnerability, data were subsequently analyzed with a hierarchical model at the sgRNA level (two-line model) and gene level (Hill curve). The vulnerability quantification is shown below. [More information]

M. tuberculosis H37Rv Reference Fitness Experiment M. tuberculosis HN878 Reference Fitness Experiment M. smegmatis Reference Fitness Experiment
 
 
M. tuberculosis H37Rv Gene-Level Logistic Regression M. tuberculosis HN878 Gene-Level Logistic Regression M. smegmatis Gene-Level Logistic Regression (Most similar ortholog)
 
Vulnerability Index (VI) -0.2030 -0.8480 -0.0530
VI Lower Bound -2.2540 -3.0540 -0.1250
VI Upper Bound 2.9350 2.2700 0.0240
Chemical Genetics

Experiment: To define genes that control drug potency in Mtb, we performed 90 CRISPRi screens across nine drugs in the Mtb reference strain, H37Rv. These screens used a genome-scale CRISPRi library (RLC12) containing 96,700 sgRNAs to enable titratable knockdown for nearly all Mtb genes. Anhydrotetracycline (ATc) was added 1 or 5 days prior to drug exposure to transcriptionally activate CRISPRi and deplete target gene products. Drugs were screened at concentrations spanning the predicted minimum inhibitory concentration (MIC). Triplicate CRISPRi library cultures were outgrown for two weeks in the presence or absence of drug. After outgrowth, we harvested genomic DNA from cultures treated with three descending doses of partially inhibitory drug concentrations (High, Med, and Low) and analyzed sgRNA abundance by deep sequencing. Hits were identified by MAGeCK as those genes whose CRISPRi inhibition reduced or increased relative fitness in the presence of a given drug (false discovery rate (FDR) < 0.01, log2 fold-change |L2FC| > 1).

Shown below are the feature-expression heatmaps for this gene for the indicated drugs from the 1-day and 5-day CRISPRi library pre-depletion screens. The black triangles denote increasing drug dose (Low, Med, High). The color of each circle represents the gene-level L2FC. A white dot represents an FDR < 0.01 and a |L2FC| > 1. If the FDR for this gene under a given drug screening condition was greater than 0.01, no L2FC value is plotted.

You can click on each square in the feature-expression heatmap to display this gene's behavior in the presence of the indicated drug at the indicated dose in two volcano plots. The “sgRNA level” volcano plot shows the L2FC value and FDR for each sgRNA targeting the gene of interest. Only sgRNAs above the sequencing limit of detection (counts > 100 after normalizing for sequencing depth) are displayed. sgRNAs are color coded according to predicted strength (see Bosch & DeJesus et al. for details, PMID: 34297925). The “Gene level” volcano plot shows the L2FC value and FDR for each gene, with the gene of interest marked with a large green dot.

1 day predepletion BDQ 1D Low BDQ 1D Med BDQ 1D High CLR 1D Low CLR 1D Med CLR 1D High EMB 1D Low EMB 1D Med EMB 1D High INH 1D Low INH 1D Med INH 1D High LVX 1D Low LVX 1D Med LVX 1D High LZD 1D Low LZD 1D Med LZD 1D High RIF 1D Low RIF 1D Med RIF 1D High STR 1D Low STR 1D Med STR 1D High VAN 1D Low VAN 1D Med VAN 1D High
5 day predepletion BDQ 5D Low BDQ 5D Med BDQ 5D High CLR 5D Low CLR 5D Med CLR 5D High EMB 5D Low EMB 5D Med EMB 5D High INH 5D Low INH 5D Med INH 5D High LVX 5D Low LVX 5D Med LVX 5D High LZD 5D Low LZD 5D Med LZD 5D High RIF 5D Low RIF 5D Med RIF 5D High STR 5D Low STR 5D Med STR 5D High VAN 5D Low VAN 5D Med VAN 5D High

Specific References

General References