Green Fluorescent Protein as a protein localization and topological reporter in mycobacteria

Belardinelli, Juán Manuel; Jackson, Mary

doi:10.1016/j.tube.2017.04.001

Cited by 13 publications

(9 citation statements)

References 16 publications

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“…Plasmids expressing PE fused at the C terminus with either the GFP or the alkaline phosphatase (PhoA) (Table S1) were introduced in the WT strain of M. smegmatis, and the GFP and PhoA activities were monitored to determine the subcellular localization of the reporter proteins. GFP exhibits fluorescence only when localized to the cytosolic compartment, whereas PhoA is only active inside the periplasmic space (15,16). Expression of PE-GFP or PE-PhoA in an M. smegmatis pE knockout mutant (PMM229) restored TPP production, indicating that the fusion proteins are correctly folded and display proper subcellular localization in bacteria (Fig.…”

Section: Resultsmentioning

confidence: 94%

The final assembly of trehalose polyphleates takes place within the outer layer of the mycobacterial cell envelope

Thouvenel

Prevot

Chiaradia

et al. 2020

Journal of Biological Chemistry

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Trehalose polyphleates (TPP) are high-molecular-weight, surface-exposed glycolipids present in a broad range of nontuberculous mycobacteria. These compounds consist of a trehalose core bearing polyunsaturated fatty acyl substituents (called phleic acids) and a straight-chain fatty acid residue, and share a common basic structure with trehalose-based glycolipids produced by Mycobacterium tuberculosis. TPP production starts in the cytosol with the formation of a diacyltrehalose intermediate. An acyltransferase, called PE, subsequently catalyzes the transfer of phleic acids onto diacyltrehalose to form TPP, and an MmpL transporter promotes the export of TPP or its precursor across the plasma membrane. PE is predicted to be an anchored membrane protein, but its topological organization is unknown, raising questions about the subcellular localization of the final stage of TPP biosynthesis and the chemical nature of the substrates that are translocated by the MmpL transporter. Here, using genetic, biochemical, and proteomic approaches, we established that PE of Mycobacterium smegmatis is exported to the cell envelope following cleavage of its signal peptide and that this process is required for TPP biosynthesis, indicating that the last step of TPP formation occurs in the outer layers of the mycobacterial cell envelope. These results provide detailed insights into the molecular mechanisms controlling TPP formation and transport to the cell surface, enabling us to propose an updated model of the TPP biosynthetic pathway. Because the molecular mechanisms of glycolipid production are conserved among mycobacteria, these findings obtained with PE from M. smegmatis may offer clues to glycolipid formation in M. tuberculosis.

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Section: Resultsmentioning

confidence: 94%

The final assembly of trehalose polyphleates takes place within the outer layer of the mycobacterial cell envelope

Thouvenel

Prevot

Chiaradia

et al. 2020

Journal of Biological Chemistry

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“…To go further, the dynamic location of LipG within living cells was investigated by using fluorescence microscopy [ 66 ]. Gene fusions between Rv0646c or MSMEG_1352 and the sfGFP gene encoding the sfGFP [ 67 ] were generated and used to transform M. smegmatis cells.…”

Section: Resultsmentioning

confidence: 99%

“…However, bioinformatics analysis did not reveal the presence of any secretion signals or specific motifs, which may indicate that the protein is surface exposed. Herein, using different cell-fractionation approaches, we demonstrated that LipG MTB is associated with the membrane fraction [ 54 , 55 , 66 ], but not translocated across the cytoplasmic membrane and remains a cytoplasmic protein which is mainly peripheral ( Figure 4 ). It has been described that such peripheral proteins require either a lipophilic attachment (i.e.…”

Section: Discussionmentioning

confidence: 99%

LipG a bifunctional phospholipase/thioesterase involved in mycobacterial envelope remodeling

et al. 2018

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Tuberculosis caused by Mycobacterium tuberculosis is currently one of the leading causes of death from an infectious agent. The main difficulties encountered in eradicating this bacteria are mainly related to (i) a very complex lipid composition of the bacillus cell wall, (ii) its ability to hide from the immune system inside the granulomas, and (iii) the increasing number of resistant strains. In this context, we were interested in the Rv0646c (lipGMTB) gene located upstream to the mmaA cluster which is described as being crucial for the production of cell wall components and required for the bacilli adaptation and survival in mouse macrophages. Using biochemical experiments combined with the construction of deletion and overexpression mutant strains in Mycobacterium smegmatis, we found that LipGMTB is a cytoplasmic membrane-associated enzyme that displays both phospholipase and thioesterase activities. Overproduction of LipGMTB decreases the glycopeptidolipids (GPL) level concomitantly to an increase in phosphatidylinositol (PI) which is the precursor of the PI mannoside (PIM), an essential lipid component of the bacterial cell wall. Conversely, deletion of the lipGMS gene in M. smegmatis leads to an overproduction of GPL, and subsequently decreases the strain susceptibility to various antibiotics. All these findings demonstrate that LipG is involved in cell envelope biosynthesis/remodeling, and consequently this enzyme may thus play an important role in mycobacterial physiology.

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“…Among other applications, these FPs have been used in biosensors systems to monitor intracellular physiological parameters, like the pH, the oxygen level or the antioxidant activity (Potzkei et al, 2012; Crone et al, 2013; Germond et al, 2016). They are also useful as reporters of gene expression and transcription regulation (Chalfie et al, 1994; Webb et al, 1995; Ruiz-Cruz et al, 2010; Uliczka et al, 2011; Mohedano et al, 2014), or as biomarkers for imaging the subcellular location of fusion proteins and their targets (Webb et al, 1995; Phillips, 2001; Belardinelli and Jackson, 2017; Kapanidis et al, 2018), for direct detection and quantification of plasmid conjugation and plasmid loss (Nancharaiah et al, 2003; Sørensen et al, 2003; Bahl et al, 2004; Karimi et al, 2016), and for tracking the bacterial cells in in vivo and in vitro competition and interactomic studies (Singer et al, 2010; Campbell-Valois and Sansonetti, 2014; Jarchum, 2015; Russo et al, 2015). Imaging using these FPs as biomarkers is especially useful in living cells because is a non-destructive and minimally invasive method that does not require exogenously added substrates or cofactors (Chalfie et al, 1994).…”

Section: Introductionmentioning

confidence: 99%

Combining Modules for Versatile and Optimal Labeling of Lactic Acid Bacteria: Two pMV158-Family Promiscuous Replicons, a Pneumococcal System for Constitutive or Inducible Gene Expression, and Two Fluorescent Proteins

et al. 2019

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Labeling of bacterial cells with fluorescent proteins allows tracking the bacteria in competition and interactomic in vivo and in vitro studies. During the last years, a few plasmid vectors have been developed aimed at the fluorescent labeling of specific members of the lactic acid bacteria (LAB), a heterogeneous group that includes microorganisms used in the food industry, as probiotics, or as live vectors for mucosal vaccines. Successful and versatile labeling of a broad range of LAB not only requires a vector containing a promiscuous replicon and a widely recognized expression system for the constitutive or regulated expression of the fluorescence determinant, but also the knowledge of the main features of the entire plasmid/host/fluorescent protein ensemble. By using the LAB model species Lactococcus lactis , we have compared the utility properties of a set of labeling vectors constructed by combining a promiscuous replicon (pMV158 or pSH71) of the pMV158 plasmid family with the gene encoding either the EGFP or the mCherry fluorescent protein placed under control of promoter P X or P M from the pneumococcal mal gene cluster for maltosaccharide uptake and utilization, respectively. Some vectors carrying P M also harbor the malR gene, whose product represses transcription from this promoter, thus enabling maltose-inducible synthesis of the fluorescent proteins. We have determined the plasmid copy number (PCN) and segregational stability of the different constructs, as well as the effect of these features on the fitness and fluorescence intensity of the lactococcal host. Constructs based on the pSH71 replicon had a high copy number (∼115) and were segregationally stable. The copy number of vectors based on the pMV158 replicon was lower (∼8–45) and varied substantially depending on the genetic context of the plasmid and on the bacterial growth conditions; as a consequence, inheritance of these vectors was less stable. Synthesis of the fluorescent proteins encoded by these plasmids did not significantly decrease the host fitness. By employing inducible expression vectors, the fluorescent proteins were shown to be very stable in this bacterium. Importantly, conditions for accurate quantification of the emitted fluorescence were established based on the maturation times of the fluorescent proteins.

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Green Fluorescent Protein as a protein localization and topological reporter in mycobacteria

Cited by 13 publications

References 16 publications

The final assembly of trehalose polyphleates takes place within the outer layer of the mycobacterial cell envelope

The final assembly of trehalose polyphleates takes place within the outer layer of the mycobacterial cell envelope

LipG a bifunctional phospholipase/thioesterase involved in mycobacterial envelope remodeling

Combining Modules for Versatile and Optimal Labeling of Lactic Acid Bacteria: Two pMV158-Family Promiscuous Replicons, a Pneumococcal System for Constitutive or Inducible Gene Expression, and Two Fluorescent Proteins

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