Inactivation of MSMEG_0412 gene drastically affects surface related properties of Mycobacterium smegmatis

Migliardi, Adriana; D’Alonzo, Daniele; Lombardi, Angela; Varcamonti, Mario; Cordone, Angelina

doi:10.1186/s12866-016-0888-z

Cited by 12 publications

(10 citation statements)

References 36 publications

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“…The bound dye was solubilized in acetic acid, and the eluted stain was quantified by recording the absorbance at 595 nm. For the sliding motility assay, the protocol was adapted essentially as described previously (59). The degree of spread was determined by measuring the diameter of the growth zone.…”

Section: Methodsmentioning

confidence: 99%

Insights into the Physiology and Metabolism of a Mycobacterial Cell in an Energy-Compromised State

Patil

Jain

2019

J Bacteriol

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Mycobacterium tuberculosis, a bacterium that causes tuberculosis, poses a serious threat, especially due to the emergence of drug-resistant strains. M. tuberculosis and other mycobacterial species, such as M. smegmatis, are known to generate an inadequate amount of energy by substrate-level phosphorylation and mandatorily require oxidative phosphorylation (OXPHOS) for their growth and metabolism. Hence, antibacterial drugs, such as bedaquiline, targeting the multisubunit ATP synthase complex, which is required for OXPHOS, have been developed with the aim of eliminating pathogenic mycobacteria. Here, we explored the influence of suboptimal OXPHOS on the physiology and metabolism of M. smegmatis. M. smegmatis harbors two identical copies of atpD, which codes for the β subunit of ATP synthase. We show that upon deletion of one copy of atpD (M. smegmatis ΔatpD), M. smegmatis synthesizes smaller amounts of ATP and enters into an energy-compromised state. The mutant displays remarkable phenotypic and physiological differences from the wild type, such as respiratory slowdown, reduced biofilm formation, lesser amounts of cell envelope polar lipids, and increased antibiotic sensitivity compared to the wild type. Additionally, M. smegmatis ΔatpD overexpresses genes belonging to the dormancy operon, the β-oxidation pathway, and the glyoxylate shunt, suggesting that the mutant adapts to a low energy state by switching to alternative pathways to produce energy. Interestingly, M. smegmatis ΔatpD shows significant phenotypic, metabolic, and physiological similarities with bedaquiline-treated wild-type M. smegmatis. We believe that the identification and characterization of key metabolic pathways functioning during an energy-compromised state will enhance our understanding of bacterial adaptation and survival and will open newer avenues in the form of drug targets that may be used in the treatment of mycobacterial infections. IMPORTANCE M. smegmatis generates an inadequate amount of energy by substrate-level phosphorylation and mandatorily requires oxidative phosphorylation (OXPHOS) for its growth and metabolism. Here, we explored the influence of suboptimal OXPHOS on M. smegmatis physiology and metabolism. M. smegmatis harbors two identical copies of the atpD gene, which codes for the ATP synthase β subunit. Here, we carried out the deletion of only one copy of atpD in M. smegmatis to understand the bacterial survival response in an energy-deprived state. M. smegmatis ΔatpD shows remarkable phenotypic, metabolic, and physiological differences from the wild type. Our study thus establishes M. smegmatis ΔatpD as an energy-compromised mycobacterial strain, highlights the importance of ATP synthase in mycobacterial physiology, and further paves the way for the identification of novel antimycobacterial drug targets.

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Section: Methodsmentioning

confidence: 99%

Insights into the Physiology and Metabolism of a Mycobacterial Cell in an Energy-Compromised State

Patil

Jain

2019

J Bacteriol

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“…The outermost layer of mycobacterial cell wall is rich in extractable noncovalently linked apolar lipids and polar glycolipids, such as glycopeptidolipids (GPLs). The GPLs are the major surface exposed molecules found in various mycobacterial species, and are required for cell aggregation . To better understand the above cell wall variations after the augmentation of pbpB , the alterations of polar and apolar lipids in the cell wall were analyzed.…”

Section: Resultsmentioning

confidence: 99%

Enhanced conversion of sterols to steroid synthons by augmenting the peptidoglycan synthesis gene pbpB in Mycobacterium neoaurum

Sun

Liu

et al. 2019

J Basic Microbiol

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Some species of mycobacteria have been modified to transform sterols to valuable steroid synthons. The unique cell wall of mycobacteria has been recognized as an important organelle to absorb sterols. Some cell wall inhibitors (e.g., vancomycin and glycine) have been validated to enhance sterol conversion by interfering with transpeptidation in peptidoglycan biosynthesis. Therefore, two transpeptidase genes, pbpA and pbpB, were selected to rationally modify the cell wall to simulate the enhancement effect of vancomycin and glycine on sterol conversion in a 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) producing strain (WIII). Unexpectedly, the pbpA or pbpB gene augmentation was conducive to the utilization of sterols. The pbpB augmentation strain WIII-pbpB was further investigated for its better performance. Compared to WIII, the morphology of WIII-pbpB was markedly changed from oval to spindle, indicating alterations of the cell wall. Biochemical analysis indicated that the altered cell wall properties of WIII-pbpB might contribute to the positive effect on sterol utilization. The productivity of 4-HBC was enhanced by 28% in the WIII-pbpB strain compared to that of WIII. These results demonstrated that the modification of peptidoglycan synthesis can improve the conversion of sterols to steroid synthons in mycobacteria.

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“…Biofilm formation was tested by growing bacterial cells in 24-well culture plates containing LB medium for Bacillus licheniformis and Sauton medium (0.05% KH 2 PO 4 , 0.05% MgSO 4 7H 2 O, 0.2% citric acid, 0.005% ferric ammonium citrate, 6% glycerol, 0.4% Asparagine, 0.05% Tween 80, pH 7.4) for Mycobacterium smegmatis [ 55 ] and incubated in a static condition at 37 °C for 48 h and 1 week, respectively. For pre-exposure experiments, cell-free supernatants of SF106 and SF174 were added to the wells together with the bacterial target strains while for post-exposure experiments the supernatant was added on the pre-formed biofilm and incubated for 24 h at 37 °C.…”

Section: Methodsmentioning

confidence: 99%

Comparative Genomics and Physiological Characterization of Two Aerobic Spore Formers Isolated from Human Ileal Samples

Saggese

Giglio²,

D’Anzi³

et al. 2022

IJMS

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Spore formers are ubiquitous microorganisms commonly isolated from most environments, including the gastro-intestinal tract (GIT) of insects and animals. Spores ingested as food and water contaminants safely transit the stomach and reach the intestine, where some of them germinate and temporarily colonize that niche. In the lower part of the GIT, they re-sporulate and leave the body as spores, therefore passing through their entire life cycle in the animal body. In the intestine, both un-germinated spores and germination-derived cells interact with intestinal and immune cells and have health-beneficial effects, which include the production of useful compounds, protection against pathogenic microorganisms, contribution to the development of an efficient immune system and modulation of the gut microbial composition. We report a genomic and physiological characterization of SF106 and SF174, two aerobic spore former strains previously isolated from ileal biopsies of healthy human volunteers. SF106 and SF174 belong respectively to the B. subtilis and Alkalihalobacillus clausii (formerly Bacillus clausii) species, are unable to produce toxins or other metabolites with cytotoxic activity against cultured human cells, efficiently bind mucin and human epithelial cells in vitro and produce molecules with antimicrobial and antibiofilm activities.

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Inactivation of MSMEG_0412 gene drastically affects surface related properties of Mycobacterium smegmatis

Cited by 12 publications

References 36 publications

Insights into the Physiology and Metabolism of a Mycobacterial Cell in an Energy-Compromised State

Insights into the Physiology and Metabolism of a Mycobacterial Cell in an Energy-Compromised State

Enhanced conversion of sterols to steroid synthons by augmenting the peptidoglycan synthesis gene pbpB in Mycobacterium neoaurum

Comparative Genomics and Physiological Characterization of Two Aerobic Spore Formers Isolated from Human Ileal Samples

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