Structural analysis and functional study of phosphofructokinase B (PfkB) from Mycobacterium marinum

Gao, Baocai; Ji, Rui; Li, Zhengyang; Su, Xiaoyu; Li, Hongyong; Sun, Yicheng; Ji, Chaoneng; Gan, Jianhua; Li, Jixi

doi:10.1016/j.bbrc.2021.09.051

Cited by 3 publications

(2 citation statements)

References 35 publications

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“…FA is involved in ribosome and other metabolic pathways, indirectly indicating the key role of folA gene in aminoacyl-tRNA biosynthesis pathway. Phosphofructokinase B (PfkB) belongs to the ribokinases family that uses phosphorylated sugars as substrates and catalyzes the conversion of fructose-6-phosphate to fructose-1,6-bisphosphate ( Gao et al, 2021 ). Expression of the pfkB family carbohydrate kinase genes was upregulated in ΔFolA , suggesting possible involvement in glycolysis and the pentose phosphate pathway, as well as in supporting processes such as cell growth and differentiation, and the immune response.…”

Section: Discussionmentioning

confidence: 99%

Effect of folA gene in human breast milk-derived Limosilactobacillus reuteri on its folate biosynthesis

Jiang,

Li,

Zhang

et al. 2024

Front. Microbiol.

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IntroductionFolate supplementation is crucial for the human body, and the chemically synthesized folic acid might have undesirable side effects. The use of molecular breeding methods to modify the genes related to the biosynthesis of folate by probiotics to increase folate production is currently a focus of research.MethodsIn this study, the folate-producing strain of Limosilactobacillus reuteri B1-28 was isolated from human breast milk, and the difference between B1-28 and folA gene deletion strain ΔFolA was investigated by phenotyping, in vitro probiotic evaluation, metabolism and transcriptome analysis.ResultsThe results showed that the folate producted by the ΔFolA was 2–3 folds that of the B1-28. Scanning electron microscope showed that ΔFolA had rougher surface, and the acid-producing capacity (p = 0.0008) and adhesion properties (p = 0.0096) were significantly enhanced than B1-28. Transcriptomic analysis revealed that differentially expressed genes were mainly involved in three pathways, among which the biosynthesis of ribosome and aminoacyl-tRNA occurred in the key metabolic pathways. Metabolomics analysis showed that folA affected 5 metabolic pathways, involving 89 different metabolites.DiscussionIn conclusion, the editing of a key gene of folA in folate biosynthesis pathway provides a feasible pathway to improve folate biosynthesis in breast milk-derived probiotics.

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Section: Discussionmentioning

confidence: 99%

Effect of folA gene in human breast milk-derived Limosilactobacillus reuteri on its folate biosynthesis

Jiang,

Li,

Zhang

et al. 2024

Front. Microbiol.

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“…The expression and purification processes were similar to previous works (Chen et al, 2020;Gao et al, 2021). In brief, Mtb-MazG was expressed in Escherichia coli BL21(DE3) cells.…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

Structural analysis of the housecleaning nucleoside triphosphate pyrophosphohydrolase MazG from Mycobacterium tuberculosis

et al. 2023

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The housecleaning enzyme of Mycobacterium tuberculosis (Mtb), MazG, is a nucleoside triphosphate pyrophosphohydrolase (NTP-PPase) and can hydrolyze all canonical or non-canonical NTPs into NMPs and pyrophosphate. The Mycobacterium tuberculosis MazG (Mtb-MazG) contributes to antibiotic resistance in response to oxidative or nitrosative stress under dormancy, making it a promising target for treating TB in latent infection patients. However, the structural basis of Mtb-MazG is not clear. Here we describe the crystal structure of Mtb-MazG (1–185) at 2.7 Å resolution, composed of two similar folded spherical domains in tandem. Unlike other all-α NTP pyrophosphatases, Mtb-MazG has an N-terminal extra region composed of three α-helices and five β-strands. The second domain is global, with five α-helices located in the N-terminal domain. Gel-filtration assay and SAXS analysis show that Mtb-MazG forms an enzyme-active dimer in solution. In addition, the metal ion Mg2+ is bound with four negative-charged residues Glu119, Glu122, Glu138, and Asp141. Different truncations and site-directed mutagenesis revealed that the full-length dimeric form and the metal ion Mg2+ are indispensable for the catalytic activity of Mtb-MazG. Thus, our work provides new insights into understanding the molecular basis of Mtb-MazG.

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A CRISPR-Nonhomologous End-Joining-based strategy for rapid and efficient gene disruption inMycobacterium abscessus

Zeng,

Ju,

Alam

et al. 2024

Preprint

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Mycobacterium abscessus, a fast-growing, non-tuberculous mycobacterium resistant to most antimicrobial drugs, poses a significant public health challenge because of its ability to cause many types of serious infections in humans. While genetic manipulation tools forM. abscessusare still being developed, the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) systems have shown promise in generating highly specific double-strand breaks (DSBs) in its genome. These DSBs can be repaired by the error-prone nonhomologous end joining (NHEJ) mechanism, facilitating targeted gene editing. Here, our study marks a pioneering application of the CRISPR-NHEJ strategy inM. abscessus. Moreover, our research uncovered an unexpected finding: contrary to previous observations inMycobacterium tuberculosisand other species, the inhibition of RecA or disruption of key genes in the homologous recombination or single-strand annealing pathways resulted in a significant decrease in NHEJ repair efficiency inM. abscessus. This discovery challenges the established perspectives and offers new insights into the interaction among the three DSB repair pathways in Mycobacterium species.IMPORTANCEThere are still very few genetic manipulation tools available forMycobacterium abscessus.. Here we report the successful application of CRISPR-Cas12a-assisted nonhomologous end joining (NHEJ) in efficient gene editing inM. abscessus. Contrary to previous research suggesting that homologous recombination (HR) inhibition may enhance such editing efficiency in other Mycobacterium species, our results showed that disruption of either HR or single-strand annealing (SSA) DNA double-strand breaks (DSBs) repair pathways not only failed to enhance but also significantly reduced the gene editing efficiency inM. abscessus. This suggests that NHEJ repair inM. abscessusmay require components from both HR and SSA pathways, highlighting a complex interaction among the DSB repair pathways inM. abscessus.

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Structural analysis and functional study of phosphofructokinase B (PfkB) from Mycobacterium marinum

Cited by 3 publications

References 35 publications

Effect of folA gene in human breast milk-derived Limosilactobacillus reuteri on its folate biosynthesis

Effect of folA gene in human breast milk-derived Limosilactobacillus reuteri on its folate biosynthesis

Structural analysis of the housecleaning nucleoside triphosphate pyrophosphohydrolase MazG from Mycobacterium tuberculosis

A CRISPR-Nonhomologous End-Joining-based strategy for rapid and efficient gene disruption inMycobacterium abscessus

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