scholarly journals Subcellular Localization of a Small Sporulation Protein in Bacillus subtilis

2003 ◽  
Vol 185 (4) ◽  
pp. 1391-1398 ◽  
Author(s):  
Christiaan van Ooij ◽  
Richard Losick
Keyword(s):  
Bacillus Subtilis ◽  
Cell Membrane ◽  
Subcellular Localization ◽  
Mother Cell ◽  
Green Fluorescence Protein ◽  
Protein S ◽  
Anchoring Protein ◽  
Unknown Gene ◽  
Fluorescence Protein ◽  
Α Helix

ABSTRACT SpoVM is an unusually small (26-residue-long) protein that is produced in the mother cell chamber of the sporangium during the process of sporulation in Bacillus subtilis. We investigated the subcellular localization of SpoVM, which is believed to be an amphipathic α-helix, by using a fusion of the sporulation protein to the green fluorescence protein (GFP). We found that SpoVM-GFP is recruited to the polar septum shortly after the sporangium undergoes asymmetric division and that the fusion protein localizes to the mother cell membrane that surrounds the forespore during the subsequent process of engulfment. We identified a patch of three residues near the N terminus of the proposed α-helix that is needed both for proper subcellular localization and for SpoVM function. We also identified a patch of residues on the opposite face of the helix and residues near both ends of the protein that are needed for SpoVM function but not for subcellular localization. Subcellular localization of SpoVM-GFP was found to require an unknown gene(s) under the control of the mother cell transcription factor σE. We propose that the N-terminal patch binds to an unknown anchoring protein that is produced under the control of σE and that other residues important in SpoVM function to recruit an unknown sporulation protein(s) to the mother cell membrane that surrounds the forespore. Our results provide evidence that SpoVM function depends on proper subcellular localization.

scholarly journals Forespore Expression and Processing of the SigE Transcription Factor in Wild-Type and Mutant Bacillus subtilis

1998 ◽  
Vol 180 (7) ◽  
pp. 1673-1681 ◽  
Author(s):  
Jingliang Ju ◽  
Tingqiu Luo ◽  
W. G. Haldenwang
Keyword(s):  
Transcription Factor ◽  
Bacillus Subtilis ◽  
Green Fluorescence Protein ◽  
Distal Portion ◽  
Protein Product ◽  
Wild Type ◽  
Green Fluorescence ◽  
Selective Degradation ◽  
Fluorescence Protein ◽  
A Site

ABSTRACT ςE is a mother cell-specific transcription factor of sporulating Bacillus subtilis that is derived from an inactive precursor protein (pro-ςE). To examine the process that prevents ςE activity from developing in the forespore, we fused the ςE structural gene (sigE) to forespore-specific promoters (PdacF and PspoIIIG ), placed these fusions at sites on the B. subtilis chromosome which translocate into the forespore either early or late, and used Western blot analysis to monitor SigE accumulation and pro-ςE processing. sigE alleles, placed at sites which entered the forespore early, were found to generate more protein product than the same fusion placed at a late entering site. SigE accumulation and processing in the forespore were enhanced by null mutations in spoIIIE, a gene whose product is essential for translocation of the distal portion of the B. subtilischromosome into the forespore. In other experiments, a chimera of pro-ςE and green fluorescence protein, previously shown to be unprocessed if it is synthesized within the forespore, was found to be processed in this compartment if coexpressed with the gene for the pro-ςE-processing enzyme, SpoIIGA. The need forspoIIGA coexpression is obviated in the absence of SpoIIIE. We interpret these results as evidence that selective degradation of both SigE and SpoIIGA prevent mature ςE from accumulating in the forespore compartment of wild-type B. subtilis. Presumably, a gene(s) located at a site that is distal to the origin of chromosome transfer is responsible for this phenomenon when it is translocated and expressed in the forespore.

scholarly journals Correlative Localization Analysis Between mRNA and Enhanced Green Fluorescence Protein-Fused Protein by a Single-Molecule Fluorescence in situ Hybridization Using an egfp Probe in Aspergillus oryzae

2021 ◽  
Vol 2 ◽  
Author(s):  
Yuki Morita ◽  
Yoshinori Katakura ◽  
Kaoru Takegawa ◽  
Yujiro Higuchi
Keyword(s):  
Subcellular Localization ◽  
Aspergillus Oryzae ◽  
Single Molecule ◽  
Green Fluorescence Protein ◽  
Green Fluorescence ◽  
Enhanced Green Fluorescence Protein ◽  
Single Molecule Fluorescence ◽  
Localization Analysis ◽  
Fluorescence Protein

Although subcellular localization analysis of proteins fused with enhanced green fluorescence protein (EGFP) has been widely conducted in filamentous fungi, little is known about the localization of messenger RNAs (mRNAs) encoding the EGFP-fused proteins. In this study, we performed single-molecule fluorescence in situ hybridization (smFISH) using an egfp probe to simultaneously visualize EGFP-fused proteins and their mRNAs in the hyphal cells of the filamentous fungus Aspergillus oryzae. We investigated the subcellular localization of mRNAs encoding cytoplasmic EGFP, an actin marker protein Lifeact tagged with EGFP, and several EGFP-fused proteins AoSec22, AoSnc1, AoVam3, and AoUapC that localize to the endoplasmic reticulum (ER), the apical vesicle cluster Spitzenkörper, vacuolar membrane, and plasma membrane, respectively. Visualization of these mRNAs by smFISH demonstrated that each mRNA exhibited distinct localization patterns likely depending on the mRNA sequence. In particular, we revealed that mRNAs encoding Lifeact-EGFP, EGFP-AoSec22, EGFP-AoVam3, and AoUapC-EGFP, but not cytoplasmic EGFP and EGFP-AoSnc1, were preferentially localized at the apical cell, suggesting certain mechanisms to regulate the existence of these transcripts among hyphal regions. Our findings provide the distinct localization information of each mRNA in the hyphal cells of A. oryzae.

scholarly journals Expansion of EasyClone-MarkerFree toolkit for Saccharomyces cerevisiae genome with new integration sites

2021 ◽  
Author(s):  
Mahsa Babaei ◽  
Luisa Sartori ◽  
Alexey Karpukhin ◽  
Dmitrii Abashkin ◽  
Elena Matrosova ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Recombinant Dna ◽  
Green Fluorescence Protein ◽  
Cellular Growth ◽  
Green Fluorescence ◽  
Biotechnological Production ◽  
Yeast Saccharomyces Cerevisiae ◽  
Integration Sites ◽  
Fluorescence Protein ◽  
Integration Efficiency

Abstract Biotechnological production requires genetically stable recombinant strains. To ensure genomic stability, recombinant DNA is commonly integrated into the genome of the host strain. Multiple genetic tools have been developed for genomic integration into baker's yeast Saccharomyces cerevisiae. Previously, we had developed a vector toolkit EasyClone-MarkerFree for stable integration into eleven sites on chromosomes X, XI, and XII of S. cerevisiae. The markerless integration was enabled by CRISPR-Cas9 system. In this study, we have expanded the kit with eight additional intergenic integration sites located on different chromosomes. The integration efficiency into the new sites was above 80%. The expression level of green fluorescence protein (gfp) for all eight sites was similar or above XI-2 site from the original EasyClone-MarkerFree toolkit. The cellular growth was not affected by the integration into any of the new eight locations. The eight-vector expansion kit is available from AddGene.

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