scholarly journals Benchmarking Various Green Fluorescent Protein Variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for Live Cell Imaging

2013 ◽  
Vol 79 (20) ◽  
pp. 6481-6490 ◽  
Author(s):  
Wout Overkamp ◽  
Katrin Beilharz ◽  
Ruud Detert Oude Weme ◽  
Ana Solopova ◽  
Harma Karsens ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Green Fluorescent Protein ◽  
Streptococcus Pneumoniae ◽  
Lactococcus Lactis ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Experimental Conditions ◽  
Content Type ◽  
Green Fluorescent

ABSTRACTGreen fluorescent protein (GFP) offers efficient ways of visualizing promoter activity and protein localizationin vivo, and many different variants are currently available to study bacterial cell biology. Which of these variants is best suited for a certain bacterial strain, goal, or experimental condition is not clear. Here, we have designed and constructed two “superfolder” GFPs with codon adaptation specifically forBacillus subtilisandStreptococcus pneumoniaeand have benchmarked them against five other previously available variants of GFP inB. subtilis,S. pneumoniae, andLactococcus lactis, using promoter-gfpfusions. Surprisingly, the best-performing GFP under our experimental conditions inB. subtiliswas the one codon optimized forS. pneumoniaeandvice versa. The data and tools described in this study will be useful for cell biology studies in low-GC-rich Gram-positive bacteria.

scholarly journals The Canonical Twin-Arginine Translocase Components Are Not Required for Secretion of Folded Green Fluorescent Protein from the Ancestral Strain of Bacillus subtilis

2014 ◽  
Vol 80 (10) ◽  
pp. 3219-3232 ◽  
Author(s):  
Anthony J. Snyder ◽  
Sampriti Mukherjee ◽  
J. Kyle Glass ◽  
Daniel B. Kearns ◽  
Suchetana Mukhopadhyay
Keyword(s):  
Bacillus Subtilis ◽  
Green Fluorescent Protein ◽  
Signal Peptide ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Secretion Systems ◽  
Content Type ◽  
Twin Arginine Translocase ◽  
Green Fluorescent ◽  
Ancestral Strain

ABSTRACTCellular processes, such as the digestion of macromolecules, phosphate acquisition, and cell motility, require bacterial secretion systems. InBacillus subtilis, the predominant protein export pathways are Sec (generalized secretory pathway) and Tat (twin-arginine translocase). Unlike Sec, which secretes unfolded proteins, the Tat machinery secretes fully folded proteins across the plasma membrane and into the medium. Proteins are directed for Tat-dependent export by N-terminal signal peptides that contain a conserved twin-arginine motif. Thus, utilizing the Tat secretion system by fusing a Tat signal peptide is an attractive strategy for the production and export of heterologous proteins. As a proof of concept, we expressed green fluorescent protein (GFP) fused to the PhoD Tat signal peptide in the laboratory and ancestral strains ofB. subtilis. Secretion of the Tat-GFP construct, as well as secretion of proteins in general, was substantially increased in the ancestral strain. Furthermore, our results show that secreted, fluorescent GFP could be purified directly from the extracellular medium. Nonetheless, export was not dependent on the known Tat secretion components or the signal peptide twin-arginine motif. We propose that the ancestral strain contains additional Tat components and/or secretion regulators that were abrogated following domestication.

scholarly journals The Origin of Chromosomal Replication Is Asymmetrically Positioned on the Mycobacterial Nucleoid, and the Timing of Its Firing Depends on HupB

2018 ◽  
Vol 200 (10) ◽  
Author(s):  
Joanna Hołówka ◽  
Damian Trojanowski ◽  
Mateusz Janczak ◽  
Dagmara Jakimowicz ◽  
Jolanta Zakrzewska-Czerwińska
Keyword(s):  
Cell Cycle ◽  
Green Fluorescent Protein ◽  
Cell Biology ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Replication Initiation ◽  
Replication Process ◽  
Content Type ◽  
Mycobacterial Cell ◽  
Green Fluorescent

ABSTRACTThe bacterial chromosome undergoes dynamic changes in response to ongoing cellular processes and adaptation to environmental conditions. Among the many proteins involved in maintaining this dynamism, the most abundant is the nucleoid-associated protein (NAP) HU. In mycobacteria, the HU homolog, HupB, possesses an additional C-terminal domain that resembles that of eukaryotic histones H1/H5. Recently, we demonstrated that the highly abundant HupB protein occupies the entirety of theMycobacterium smegmatischromosome and that the HupB-binding sites exhibit a bias from the origin (oriC) to the terminus (ter). In this study, we used HupB fused with enhanced green fluorescent protein (EGFP) to perform the first analysis of chromosome dynamics and to track theoriCand replication machinery directly on the chromosome during the mycobacterial cell cycle. We show that the chromosome is located in an off-center position that reflects the unequal division and growth of mycobacterial cells. Moreover, unlike the situation inE. coli, the sisteroriCregions ofM. smegmatismove asymmetrically along the mycobacterial nucleoid. Interestingly, in this slow-growing organism, the initiation of the next round of replication precedes the physical separation of sister chromosomes. Finally, we show that HupB is involved in the precise timing of replication initiation.IMPORTANCEAlthough our view of mycobacterial nucleoid organization has evolved considerably over time, we still know little about the dynamics of the mycobacterial nucleoid during the cell cycle. HupB is a highly abundant mycobacterial nucleoid-associated protein (NAP) with an indispensable histone-like tail. It was previously suggested as a potential target for antibiotic therapy against tuberculosis. Here, we fused HupB with enhanced green fluorescent protein (EGFP) to study the dynamics of the mycobacterial chromosome in real time and to monitor the replication process directly on the chromosome. Our results reveal that, unlike the situation inEscherichia coli, the nucleoid of an apically growing mycobacterium is positioned asymmetrically within the cell throughout the cell cycle. We show that HupB is involved in controlling the timing of replication initiation. Since tuberculosis remains a serious health problem, studies concerning mycobacterial cell biology are of great importance.

scholarly journals A Photostable Green Fluorescent Protein Variant for Analysis of Protein Localization in Candida albicans

2009 ◽  
Vol 9 (1) ◽  
pp. 224-226 ◽  
Author(s):  
Chengda Zhang ◽  
James B. Konopka
Keyword(s):  
Candida Albicans ◽  
Green Fluorescent Protein ◽  
Codon Usage ◽  
Signal Intensity ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Protein Variant ◽  
Green Fluorescent ◽  
Green Fluorescent Protein Variant

ABSTRACT Fusions to the green fluorescent protein (GFP) are an effective way to monitor protein localization. However, altered codon usage in Candida species has delayed implementation of new variants. Examination of three new GFP variants in Candida albicans showed that one has higher signal intensity and increased resistance to photobleaching.

Dynamic localization of penicillin-binding proteins during spore development in Bacillus subtilis

Microbiology ◽  
2005 ◽  
Vol 151 (3) ◽  
pp. 999-1012 ◽  
Author(s):  
Dirk-Jan Scheffers
Keyword(s):  
Bacillus Subtilis ◽  
Green Fluorescent Protein ◽  
Binding Proteins ◽  
Cytoplasmic Membrane ◽  
Fluorescent Protein ◽  
Spore Formation ◽  
The Other ◽  
Spore Development ◽  
Penicillin Binding Proteins ◽  
Green Fluorescent

During Bacillus subtilis spore formation, many membrane proteins that function in spore development localize to the prespore septum and, subsequently, to the outer prespore membrane. Recently, it was shown that the cell-division-specific penicillin-binding proteins (PBPs) 1 and 2b localize to the asymmetric prespore septum. Here, the author studied the localization of other PBPs, fused to green fluorescent protein (GFP), during spore formation. Fusions to PBPs 4, 2c, 2d, 2a, 3, H, 4b, 5, 4a, 4* and X were expressed during vegetative growth, and their localization was monitored during sporulation. Of these PBPs, 2c, 2d, 4b and 4* have been implicated as having a function in sporulation. It was found that PBP2c, 2d and X changed their localization, while the other PBPs tested were not affected. The putative endopeptidase PbpX appears to spiral out in a pattern that resembles FtsZ redistribution during sporulation, but a pbpX knockout strain had no distinguishable phenotype. PBP2c and 2d localize to the prespore septum and follow the membrane during engulfment, and so are redistributed to the prespore membrane. A similar pattern was observed when GFP–PBP2c was expressed in the mother cell from a sporulation-specific promoter. This work shows that various PBPs known to function during sporulation are redistributed from the cytoplasmic membrane to the prespore.

Green Fluorescent Protein: A Tool for Gene Expression and Cell Biology in Mycobacteria

2003 ◽  
pp. 245-260
Author(s):  
Laura E. Via ◽  
Subramanian Dhandayuthapani ◽  
Dusanka Deretic ◽  
V. Deretic
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Protein A ◽  
Green Fluorescent
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