Molecular Dissection of Phage Endolysin

Pohane, Amol Arunrao; Joshi, Himanshu; Jain, Vikas

doi:10.1074/jbc.m113.529594

Cited by 44 publications

(60 citation statements)

References 55 publications

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“…S1, followed by DNA sequencing of the amplified fragments. For complementation, the PCR-amplified atpD gene was cloned in an Escherichia coli-mycobacterium shuttle vector, pMVAcet, under the control of an acetamideinducible promoter (31) and was introduced in both the wild-type and the atpD-deleted bacteria, as detailed in the supplemental material.…”

Section: Methodsmentioning

confidence: 99%

“…We first recorded the M. smegmatis ΔatpD growth profile and compared it with that of the wild-type (WT) and the atpD-complemented (ΔatpD C ) M. smegmatis strains. Complementation was achieved by introducing the wild-type atpD gene on a plasmid, pMVAcet (31), and expressing it from an acetamide-inducible promoter. M. smegmatis ΔatpD showed a slow growth phenotype, in contrast to the WT and the ΔatpD C strains (Fig.…”

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confidence: 99%

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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%

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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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“…We had previously reported the construction of an acetamide-inducible plasmid, pMVAcet, which was used for the expression of various genes in M. smegmatis [14][15][16]. Here, we cloned various polypeptide tag-coding sequences in pMVAcet vector and further replaced the promoter with others inducible systems to prepare our plasmids ( Table 2).…”

Section: Construction Of Acetamide-inducible Plasmid With Polypeptidementioning

confidence: 99%

“…Next, the modified pMEND-Lx vector was digested with KpnI and NdeI enzymes, and the released insert carrying the tetRO promoter system was introduced in pMV261 vector at the same sites, thus giving rise to pMVTet plasmid (S1 Fig). It may be noted here that the pMV261 vector used here was modified in our laboratory to carry NdeI site [14]. To clone 6xHis, GFP, and GST tags under hsp60 and tetracyclineinducible promoters, respective tags along with kanamycin resistance marker were excised from pMA-His, pMA-GFP, and pMA-GST using NdeI and SpeI enzymes, and introduced in pMV261 and pMVTet vectors, at the same sites, resulting in six different plasmids having two promoter systems with three polypeptide tags each (Fig 2).…”

Section: Construction Of Heat-and Tetracycline-inducible Vectors Withmentioning

confidence: 99%

Construction of E. coli—Mycobacterium shuttle vectors with a variety of expression systems and polypeptide tags for gene expression in mycobacteria

2020

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Cloning and expression of a desired gene is indispensable in molecular biology studies. Expression vectors, in this regard, should offer much needed flexibility and choice of cloning strategies for both in vivo and in vitro protein expression experiments. Furthermore, availability of option to choose from various reporter tags allows one to be flexible during designing of an experiment in a more relevant manner. Thus, the need of a versatile expression system cannot be ignored. Although several different expression vectors are available for gene expression in mycobacteria, they lack the required versatility of expression and the inclusion of reporter tags. We here present the construction of a set of nine E. coli-Mycobacterium shuttle plasmids, which offer a combination of three mycobacterial promoter systems (heat shock inducible-hsp60, tetracycline-, and acetamide-inducible) along with three polypeptide tags (Green Fluorescent Protein (GFP), Glutathione S-transferase (GST) and hexahistidine tag). These vectors offer the cloning of a target gene in all the nine given vectors in parallel, thus allowing the generation of recombinant plasmids that will express the target gene from different promoters with different tags. Here, while the hexa-histidine and GST tags can be used for protein purification and pull-down experiments, the GFP-tag can be used for protein localization within the cell. Additionally, the vectors also offer the choice of positioning of the reporter tag either at the N-terminus or at the C-terminus of the expressed protein, which is achieved by cloning of the gene at any of the two blunt-end restriction enzyme sites available in the vector. We believe that these plasmids will be extremely useful in the gene expression studies in mycobacteria by offering the choices of promoters and reporters. Our work also paves the way to developing more such plasmids with other tags and promoters that may find use in mycobacterial biology.

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“…We further investigated the replication of DNA isolated from Msm cells after they were treated with Dma 10 . We performed colony PCR on Msm transformed with pMV‐NTD plasmid29 to see the effect of SAMPs on replication of genomic DNA (gDNA) and plasmid DNA. The cells were treated with Dma 10 (200 μg mL −1 ) while untreated transformed cells were used as control.…”

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Cell Penetrating Synthetic Antimicrobial Peptides (SAMPs) Exhibiting Potent and Selective Killing of Mycobacterium by Targeting Its DNA

Sharma

Pohane

Bansal

et al. 2015

Chemistry A European J

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Naturally occurring antimicrobial peptides (AMPs) are powerful defence tools to tackle pathogenic microbes. However, limited natural production and high synthetic costs in addition to poor selectivity limit large-scale use of AMPs in clinical settings. Here, we present a series of synthetic AMPs (SAMPs) that exhibit highly selective and potent killing of Mycobacterium (minimum inhibitory concentration <20 μg mL(-1)) over E. coli or mammalian cells. These SAMPs are active against rapidly multiplying as well as growth saturated Mycobacterium cultures. These SAMPs are not membrane-lytic in nature, and are readily internalized by Mycobacterium and mammalian cells; whereas in E. coli, the lipopolysaccharide layer inhibits their cellular uptake, and hence, their antibacterial action. Upon internalization, these SAMPs interact with the unprotected genomic DNA of mycobacteria, and impede DNA-dependent processes, leading to bacterial cell death.

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Molecular Dissection of Phage Endolysin

Cited by 44 publications

References 55 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

Construction of E. coli—Mycobacterium shuttle vectors with a variety of expression systems and polypeptide tags for gene expression in mycobacteria

Cell Penetrating Synthetic Antimicrobial Peptides (SAMPs) Exhibiting Potent and Selective Killing of Mycobacterium by Targeting Its DNA

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