Journal: mBio
Article Title: Genetic factors affecting storage and utilization of lipids during dormancy in Mycobacterium tuberculosis
doi: 10.1128/mbio.03208-23
Figure Lengend Snippet: Tn-seq and RNAtag-seq reveal that omamC is critical for survival during carbon starvation. ( A ) Carbon starvation-resuscitation growth scheme. A pooled M. tuberculosis H37Rv transposon library was grown in rich media [7H9, 10% oleic acid-albumin-dextrose-catalase (OADC), 0.5% glycerol, 0.05% tyloxapol] for 5 days, diluted into starvation media (7H9 salts, 0.05% tyloxapol), and cultured for 5 weeks, followed by resuscitation in rich media for 5 days. Transposon mutants present in the pool prior to carbon starvation and resuscitation cycling (input pool) and after three cycles of growth, starvation, and resuscitation performed over 4.5 months (output pool) were determined using Tn-seq. ( B ) Gene set enrichment analysis of genes (102) that were significantly [ P < 0.03 (Mann-Whitney U test); log 2 (FC) <−3.5] underrepresented in the output pool after three cycles of starvation and resuscitation. Genes are color coded based on the pathway with which they are associated with the positive control genes sigB and relA , known to be involved in carbon starvation, indicated in purple (compare Fig. S4; Table S3). ( C ) Of seven mce genes defined as hits by Tn-seq ( B ), only omamC is expressed during carbon starvation. Depicted are relative transcript levels during starvation of 298 genes whose corresponding transposon mutants were significantly changed in the starvation-resuscitation Tn-seq screen (with a threshold of P < 0.03, Mann-Whitney U test (MWU)). Differences in gene expression during carbon starvation compared to logarithmic growth were determined using RNAtag-seq and DESeq2 (R). The results of the Tn-seq screen are shown as fitness, i.e. , a function of the transposon input/output pool. The log 2 (FC) is shown in both cases. Depicted in gray are all genes that had a P -value <0.03 ( P (-log 10 ) >1.52) in the Tn-seq analysis. Carbon starvation controls sigB and relA ( , ) are shown in purple. All values are listed in Tables S4 to S6.
Article Snippet: Mtb H37Rv and derivative strains were grown at 37°C in Middlebrook 7H9 (Difco) liquid culture medium supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC), 0.5% glycerol, and 0.05% tyloxapol (referred to as growth in rich media in the text) or on Middlebrook 7H10 (Difco) solid culture medium supplemented with 10% OADC (BD Biosciences) and 0.5% glycerol.
Techniques: Cell Culture, MANN-WHITNEY, Positive Control, Expressing
![Tn-seq and RNAtag-seq reveal that omamC is critical for survival during carbon starvation. ( A ) Carbon starvation-resuscitation growth scheme. A pooled M. tuberculosis H37Rv transposon library was grown in rich media [7H9, 10% oleic acid-albumin-dextrose-catalase <t>(OADC),</t> 0.5% glycerol, 0.05% tyloxapol] for 5 days, diluted into starvation media (7H9 salts, 0.05% tyloxapol), and cultured for 5 weeks, followed by resuscitation in rich media for 5 days. Transposon mutants present in the pool prior to carbon starvation and resuscitation cycling (input pool) and after three cycles of growth, starvation, and resuscitation performed over 4.5 months (output pool) were determined using Tn-seq. ( B ) Gene set enrichment analysis of genes (102) that were significantly [ P < 0.03 (Mann-Whitney U test); log 2 (FC) <−3.5] underrepresented in the output pool after three cycles of starvation and resuscitation. Genes are color coded based on the pathway with which they are associated with the positive control genes sigB and relA , known to be involved in carbon starvation, indicated in purple (compare Fig. S4; Table S3). ( C ) Of seven mce genes defined as hits by Tn-seq ( B ), only omamC is expressed during carbon starvation. Depicted are relative transcript levels during starvation of 298 genes whose corresponding transposon mutants were significantly changed in the starvation-resuscitation Tn-seq screen (with a threshold of P < 0.03, Mann-Whitney U test (MWU)). Differences in gene expression during carbon starvation compared to logarithmic growth were determined using RNAtag-seq and DESeq2 (R). The results of the Tn-seq screen are shown as fitness, i.e. , a function of the transposon input/output pool. The log 2 (FC) is shown in both cases. Depicted in gray are all genes that had a P -value <0.03 ( P (-log 10 ) >1.52) in the Tn-seq analysis. Carbon starvation controls sigB and relA ( , ) are shown in purple. All values are listed in Tables S4 to S6.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5790/pmc10865790/pmc10865790__mbio.03208-23.f001.jpg)