Development and application of unstable GFP variants to kinetic studies of mycobacterial gene expression

Blokpoel, Marian C. J.; O'Toole, Ronan; Smeulders, Marjan J.; Williams, Huw D.

doi:10.1016/s0167-7012(03)00044-7

Cited by 39 publications

(31 citation statements)

References 25 publications

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“…GFP has often been used as a reporter for the expression and stability of expressed fusion proteins (1,4,8,39). Figure 3 shows that GFP, with LYK at the C terminus (construct 3A), was expressed and matured to an active form in M. smegmatis and that the induced fluorescence was stable over an ϳ8-h period after removal of the inducer.…”

Section: Resultsmentioning

confidence: 99%

In Vivo Inactivation of the Mycobacterial Integral Membrane Stearoyl Coenzyme A Desaturase DesA3 by a C-Terminus-Specific Degradation Process

et al. 2008

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DesA3 (Rv3229c) from Mycobacterium tuberculosis is a membrane-bound stearoyl coenzyme A ⌬ 9 desaturase that reacts with the oxidoreductase Rv3230c to produce oleic acid. This work provides evidence for a mechanism used by mycobacteria to regulate this essential enzyme activity. DesA3 expressed as a fusion with either a C-terminal His 6 or c-myc tag had consistently higher activity and stability than native DesA3 having the native C-terminal sequence of LAA, which apparently serves as a binding determinant for a mycobacterial protease/degradation system directed at DesA3. Fusion of only the last 12 residues of native DesA3 to the C terminus of green fluorescent protein (GFP) was sufficient to make GFP unstable. Furthermore, the comparable C-terminal sequence from the Mycobacterium smegmatis DesA3 homolog Msmeg_1886 also conferred instability to the GFP fusion. Systematic examination revealed that residues with charged side chains, large nonpolar side chains, or no side chain at the last two positions were most important for stabilizing the construct, while lesser effects were observed at the third-from-last position. Using these rules, a combinational substitution of the last three residues of DesA3 showed that either DKD or LEA gave the best enhancement of stability for the modified GFP in M. smegmatis. Moreover, upon mutagenesis of LAA at the C terminus in native DesA3 to either of these tripeptides, the modified enzyme had enhanced catalytic activity and stability. Since many proteases are conserved within bacterial families, it is reasonable that M. tuberculosis will use a similar C-terminal degradation system to posttranslationally regulate the activity of DesA3 and other proteins. Application of these rules to the M. tuberculosis genome revealed that ϳ10% the proteins encoded by essential genes may be susceptible to C-terminal proteolysis. Among these, an annotation is known for less than half, underscoring a general lack of understanding of proteins that have only temporal existence in a cell.

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Section: Resultsmentioning

confidence: 99%

In Vivo Inactivation of the Mycobacterial Integral Membrane Stearoyl Coenzyme A Desaturase DesA3 by a C-Terminus-Specific Degradation Process

et al. 2008

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“…The cells were harvested by centrifugation, washed once in PBS/0.05% Tween 80 and resuspended in the same buffer to an OD600 of approximately 1. Fluorescence was measured with a spectrofluorimeter using excitation and emission wavelengths of 480 and 510 nm respectively (Blokpoel et al, 2003).…”

Section: Analysis Of the Activity Of The Rel Promotermentioning

confidence: 99%

Polyphosphate kinase is involved in stress‐induced mprAB‐sigE‐rel signalling in mycobacteria

Sureka

Dey

Datta

et al. 2007

Molecular Microbiology

132

207

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SummaryPolyphosphate kinase 1 (PPK1) helps bacteria to survive under stress. The ppk1 gene of Mycobacterium tuberculosis was overexpressed in Escherichia coli and characterized. Residues R230 and F176, predicted to be present in the head domain of PPK1, were identified as residues critical for polyphosphate (polyP)-synthesizing ability and dimerization of PPK1. A ppk1 knockout mutant of Mycobacterium smegmatis was compromised in its ability to survive under long-term hypoxia. The transcription of the rel gene and the synthesis of the stringent response regulator ppGpp were impaired in the mutant and restored after complementation with ppk1 of M. tuberculosis, providing evidence that PPK1 is required for the stringent response. We present evidence that PPK1 is likely required for mprAB-sigE-rel signalling. s E regulates the transcription of rel, and we hypothesize that under conditions of stress polyP acts as a preferred donor for MprB-mediated phosphorylation of MprA facilitating transcription of the sigE gene thereby leading finally to the enhancement of the transcription of rel in M. smegmatis and M. tuberculosis. Downregulation of ppk1 led to impaired survival of M. tuberculosis in macrophages. PolyP plays a central role in the stress response of mycobacteria.

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“…Wild-type GFP has 2 excitation peaks with maxima at 395 nm (low-wave UV) and 475 nm (blue light) and emits green light with a peak wavelength of 507 nm. Enhanced GFP has been developed as a marker in many different biologic systems [11,12]. Liposomal transfection of the protein can be performed using the pEGFP-N1 vector [13].…”

Section: Introductionmentioning

confidence: 99%

New trends in tumor biology: transfection of a human hepatoblastoma cell line with green fluorescent protein

Warmann

Fuchs

Seitz

et al. 2005

Journal of Pediatric Surgery

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Development and application of unstable GFP variants to kinetic studies of mycobacterial gene expression

Cited by 39 publications

References 25 publications

In Vivo Inactivation of the Mycobacterial Integral Membrane Stearoyl Coenzyme A Desaturase DesA3 by a C-Terminus-Specific Degradation Process

In Vivo Inactivation of the Mycobacterial Integral Membrane Stearoyl Coenzyme A Desaturase DesA3 by a C-Terminus-Specific Degradation Process

Polyphosphate kinase is involved in stress‐induced mprAB‐sigE‐rel signalling in mycobacteria

New trends in tumor biology: transfection of a human hepatoblastoma cell line with green fluorescent protein

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