title: Optimization of fluorescent tools for cell biology studies in Gram-positive bacteria.
abstract: The understanding of how Gram-positive bacteria divide and ensure the correct localization of different molecular machineries, such as those involved in the synthesis of the bacterial cell surface, is crucial to design strategies to fight bacterial infections. In order to determine the correct subcellular localization of fluorescent proteins in Streptococcus pneumoniae, we have previously described tools to express derivatives of four fluorescent proteins, mCherry, Citrine, CFP and GFP, to levels that allow visualization by fluorescence microscopy, by fusing the first ten amino acids of the S. pneumoniae protein Wze (the i-tag), upstream of the fluorescent protein. Here, we report that these tools can also be used in other Gram-positive bacteria, namely Lactococcus lactis, Staphylococcus aureus and Bacillus subtilis, possibly due to optimized translation rates. Additionally, we have optimized the i-tag by testing the effect of the first ten amino acids of other pneumococcal proteins in the increased expression of the fluorescent protein Citrine. We found that manipulating the structure and stability of the 5' end of the mRNA molecule, which may influence the accessibility of the ribosome, is determinant to ensure the expression of a strong fluorescent signal.
authors.affiliation: Laboratory of Bacterial Cell Surfaces and Pathogenesis, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República, Apartado 127, 2781-901 Oeiras, Portugal.
authors.forename: Maria João
authors.initials: MJ
authors.lastname: Catalão
authors.affiliation: Laboratory of Bacterial Cell Surfaces and Pathogenesis, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República, Apartado 127, 2781-901 Oeiras, Portugal.
authors.forename: Joana
authors.initials: J
authors.lastname: Figueiredo
authors.affiliation: Laboratory of Bacterial Cell Surfaces and Pathogenesis, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República, Apartado 127, 2781-901 Oeiras, Portugal.
authors.forename: Mafalda X
authors.initials: MX
authors.lastname: Henriques
authors.affiliation: National Institute of Health, Department of Infectious Diseases, Av. Padre Cruz, 1649-016 Lisbon, Portugal.
authors.forename: João Paulo
authors.initials: JP
authors.lastname: Gomes
authors.affiliation: Laboratory of Bacterial Cell Surfaces and Pathogenesis, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República, Apartado 127, 2781-901 Oeiras, Portugal.
authors.forename: Sérgio R
authors.initials: SR
authors.lastname: Filipe
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D001665:Binding Sites; D003585:Cell Biology; D003062:Codon / Q000235:genetics; D017124:Conserved Sequence; D006094:Gram-Positive Bacteria / Q000378:metabolism*; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D008969:Molecular Sequence Data; D020871:RNA Stability / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism
chemical_list: D000596:Amino Acids; D003062:Codon; D008164:Luminescent Proteins; D012333:RNA, Messenger