We focused our attention on the anti-Staphylococcus molecule and found that it was active at a wide range of pH conditions and that its secretion was dependent on the growth phase, medium, and temperature. We report that SF214, a marine-isolated strain of Bacillus pumilus, produces at least two different molecules with antibacterial activity: a molecule smaller than 3 kDa active against Staphylococcus aureus and a molecule larger than 10 kDa active against Listeria monocytogenes. Producing antimicrobials is a common adaptive behavior shared by many microorganisms, including marine bacteria. Such specificity, together with its stability, propose this new antimicrobial as a tool for potential biotechnological applications in the fight against the dangerous food-borne pathogen L. pumilus peptide is unusual with respect to other antimicrobials both for its time of synthesis and secretion and for its strict specificity against L. The peptide showed a lytic activity against growing but not resting cells of Listeria monocytogenes and appeared extremely specific being inactive also against L. pumilus SF214 indicated that it is a peptide slightly smaller than 10 kDa, produced during the exponential phase of growth, stable at a wide range of pH conditions and resistant to various chemical treatments. Our analysis on the anti-Listeria molecule of B. The anti-Staphylococcus molecule has been previously characterized as a pumilacidin, a nonribosomally synthesized lipopetide composed of a mixture of cyclic heptapeptides linked to fatty acids of variable length. We have recently reported that SF214, a marine isolated strain of Bacillus pumilus, produces two different antimicrobials specifically active against either Staphylococcus aureus or Listeria monocytogenes. Members of the Bacillus genus produce a large variety of antimicrobial peptides including linear or cyclic lipopeptides and thiopeptides, that often have a broad spectrum of action against Gram-positive and Gram-negative bacteria. Due to independence of molecular oxygen and fluorescent signal, SNAPCd appears the most suitable candidate for live-cell imaging in C. The present study compares several commonly used fluorescent systems for application as transcriptional or translational reporters in microscopy and summarizes the limitations and key challenges for live-cell imaging of C. difficile histone-like protein, the fluorescent systems behaved similarly and did not affect the HupA overproduction phenotype. difficile cells limiting the applicability of this fluorophore as a transcriptional reporter. High levels of mCherryOpt were toxic for C. Cytosolic overexpression of the different fluorescent systems alone, using the same expression signals, showed heterogeneous expression of the fluorescent systems. difficile cells are exposed to environmental oxygen autofluorescence is enhanced. difficile cells increases during growth, independent of sigB or spo0A. We show that the intrinsic fluorescence of C. difficile cells harbouring different fluorescent systems, either expressing these separately in the cytosol or fused to the C-terminus of HupA, under defined conditions. We performed single cell analyses using fluorescence microscopy of exponentially growing C. In this study, we sought to characterize previously used fluorescent systems in C. For Clostridioides difficile, the fluorescent proteins CFPopt, mCherryOpt and phiLOV2.1, and the self-labelling tags SNAPCd and HaloTag, hereafter collectively referred as fluorescent systems, have been described to explore different cellular pathways. Since the pigment had an essential role in the cell resistance to oxidative stress conditions, we propose that within the heterogeneous population different survival strategies can be followed by the different cells.įluorescence microscopy is a valuable tool to study a broad variety of bacterial cell components and dynamics thereof. Pigment and matrix production in SF214 appear then as two developmental fates both alternative to sporulation. Only some of the pigmented cells were also able to produce a matrix. The fraction of cells producing the pigment varied in the different growth conditions and occured only in cells not devoted to sporulation. Only a subpopulation of stationary phase cells produced the pigment, indicating that the stationary culture contains a heterogeneous cell population and that pigment synthesis is a bimodal phenomenon. Pigmentation is strictly regulated and high pigment production was observed during the late stationary growth phase in a minimal medium and at growth temperatures lower than the optimum. Bacillus pumilus SF214 is a spore forming bacterium, isolated from a marine sample, able to produce a matrix and a orange-red, water soluble pigment.
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