Ant bacterial species, given the diversity in terms of morphology, physiology and metabolism among bacteria. Therefore, we have recently witnessed the development of new tools to allow cell biology studies in different pathogenic bacteria [1,2]. Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. This Gram-positive bacterium is associated with a range of infections, which can vary from simple otitis media to more complicated ones, such as pneumonia or meningitis. Infection by this important human pathogen is of particular concern in developing countries, in which pneumococcal septicemia causes 25 of all preventable deaths in children under the age of five [3]. In order to design new and more efficient Pleuromutilin supplier strategies to fight pneumococcal infections it is essential to understand how these bacteria divide or perform specific tasks important for their survival inside the host, such as the synthesis of peptidoglycan, the target of beta-lactam antibiotics which are widely used against S. pneumoniae, or the synthesis of the capsular polysaccharide, the target of several successful anti-pneumococcal vaccines. An important step to accomplish this goal is the study of the localization of proteins involved in these processes. However, fora long time, cell biology studies in S. pneumoniae were limited by the lack of appropriate tools. Localization of pneumococcal proteins involved in cell wall synthesis [4,5] and cell division [6,7,8] was initially accomplished using immunofluorescence techniques, which require cell fixation and lysis to allow access of the antibodies to the target proteins. Therefore, immunofluorescence can not be used with live cells and is prone to generate artifacts [9]. It was only recently that the first studies on the localization of proteins in live pneumococcal cells, using fluorescent Finafloxacin Protein fusions, tagged to Green Fluorescent Protein (GFP), was reported [1]. Since then, other proteins involved in processes such as cell division [10,11], cell wall synthesis [12] and capsular polysaccharide synthesis [13,14] have been localized in live pneumococcal cells. However, the variety of tools available for these studies is still limited. In this paper, we report the construction of new plasmids that expand the tools available for S. pneumoniae cell biology studies by allowing the expression of N- or C- terminal protein fusions 15857111 to different fluorescent reporters, namely mCherry, Citrine, CFP and GFP. For this purpose we have improved the expression of the various fluorescent proteins in S. pneumoniae, by introducing an upstream tag, named “i-tag”, which increases protein translation. The availability of these plasmids should greatly facilitate studies of protein localization in this important clinical pathogen.Expression of Fluorescent Proteins in S.pneumoniaeResults and Discussion Expression of mCherry, Citrine, CFP and GFP in S. pneumoniaeS. pneumoniae is a microaerophile organism and therefore can only grow in the presence of low levels of oxygen, which may impair the correct folding of GFP-like proteins that are known to require the presence of oxygen [15]. We have expressed fusions of Wze, a protein required for the regulation of the synthesis of the capsule polysaccharide [3], to four different fluorescent proteins, mCherry [16], Citrine [17], CFP [18] and GFP [19], two of which (CFP and GFP) had not been previously used in S. pneumoniae. The protein fusions Wze-CFP (BCSMH029) and Wze FP (BCSMH03.Ant bacterial species, given the diversity in terms of morphology, physiology and metabolism among bacteria. Therefore, we have recently witnessed the development of new tools to allow cell biology studies in different pathogenic bacteria [1,2]. Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. This Gram-positive bacterium is associated with a range of infections, which can vary from simple otitis media to more complicated ones, such as pneumonia or meningitis. Infection by this important human pathogen is of particular concern in developing countries, in which pneumococcal septicemia causes 25 of all preventable deaths in children under the age of five [3]. In order to design new and more efficient strategies to fight pneumococcal infections it is essential to understand how these bacteria divide or perform specific tasks important for their survival inside the host, such as the synthesis of peptidoglycan, the target of beta-lactam antibiotics which are widely used against S. pneumoniae, or the synthesis of the capsular polysaccharide, the target of several successful anti-pneumococcal vaccines. An important step to accomplish this goal is the study of the localization of proteins involved in these processes. However, fora long time, cell biology studies in S. pneumoniae were limited by the lack of appropriate tools. Localization of pneumococcal proteins involved in cell wall synthesis [4,5] and cell division [6,7,8] was initially accomplished using immunofluorescence techniques, which require cell fixation and lysis to allow access of the antibodies to the target proteins. Therefore, immunofluorescence can not be used with live cells and is prone to generate artifacts [9]. It was only recently that the first studies on the localization of proteins in live pneumococcal cells, using fluorescent protein fusions, tagged to Green Fluorescent Protein (GFP), was reported [1]. Since then, other proteins involved in processes such as cell division [10,11], cell wall synthesis [12] and capsular polysaccharide synthesis [13,14] have been localized in live pneumococcal cells. However, the variety of tools available for these studies is still limited. In this paper, we report the construction of new plasmids that expand the tools available for S. pneumoniae cell biology studies by allowing the expression of N- or C- terminal protein fusions 15857111 to different fluorescent reporters, namely mCherry, Citrine, CFP and GFP. For this purpose we have improved the expression of the various fluorescent proteins in S. pneumoniae, by introducing an upstream tag, named “i-tag”, which increases protein translation. The availability of these plasmids should greatly facilitate studies of protein localization in this important clinical pathogen.Expression of Fluorescent Proteins in S.pneumoniaeResults and Discussion Expression of mCherry, Citrine, CFP and GFP in S. pneumoniaeS. pneumoniae is a microaerophile organism and therefore can only grow in the presence of low levels of oxygen, which may impair the correct folding of GFP-like proteins that are known to require the presence of oxygen [15]. We have expressed fusions of Wze, a protein required for the regulation of the synthesis of the capsule polysaccharide [3], to four different fluorescent proteins, mCherry [16], Citrine [17], CFP [18] and GFP [19], two of which (CFP and GFP) had not been previously used in S. pneumoniae. The protein fusions Wze-CFP (BCSMH029) and Wze FP (BCSMH03.
