26The maintenance and organization of the chromosome plays an important role in the 27 development and survival of bacteria. Bacterial chromatin proteins are architectural proteins 28 that bind DNA, modulate its conformation and by doing so affect a variety of cellular 29 processes. No bacterial chromatin proteins of C. difficile have been characterized to date. 30Here, we investigate aspects of the C. difficile HupA protein, a homologue of the 31 histone-like HU proteins of Escherichia coli. HupA is a 10 kDa protein that is present as a 32homodimer in vitro and self-interacts in vivo. HupA co-localizes with the nucleoid of C. 33 difficile. It binds to the DNA without a preference for the DNA G+C content. Upon DNA 34 binding, HupA induces a conformational change in the substrate DNA in vitro and leads to 35 compaction of the chromosome in vivo. 36The present study is the first to characterize a bacterial chromatin protein in C. 37 difficile and opens the way to study the role of chromosomal organization in DNA 38 metabolism and on other cellular processes in this organism. 39 survival [24, 25]. However, in B. subtilis the HU protein HBsu is essential for cell viability, 67 likely due to the lack of functional redundancy of the HU proteins such as in E. coli [17, 23]. 68In solution, most HU proteins are found as homodimers or heterodimers and are able 69 to bind DNA through a flexible DNA binding domain. The crystal structure of the E. coli αHU-70 βHU heterodimer suggests the formation of higher order complexes at higher protein 71 concentrations [22]. Modeling of these complexes suggest HU proteins have the ability to 72 form higher-order complexes through dimer-dimer interaction and make nucleoprotein 73 filaments [22, 26, 27]. However, the physiological relevance of these is still unclear [18, 22, 74 27]. 75The flexible nature of the DNA-binding domain in HU proteins confers the ability to 76 accommodate diverse substrates. Most proteins bind with variable affinity and without strong 77 sequence specificity to both DNA and RNA [28]. Some bacterial chromatin proteins have a 78 clear preference for AT-rich regions [29][30][31] or for the presence of different structures on the 79 DNA [28, 32]. 80 HU proteins can modulate DNA topology in various ways. They can stabilize 81 negatively supercoiled DNA or constrain negative supercoils in the presence of 82 topoisomerase [22, 33]. HU proteins are involved in modulation of the chromosome 83 conformation and have been shown to compact DNA [16, 26, 34]. This compaction of DNA is 84 possible through the ability of HU proteins to introduce flexible hinges and/or bend the DNA 85 [16, 26, 34, 35]. 86The ability to induce conformational changes in the DNA influences a variety of 87 cellular processes due to an indirect effect on global gene expression [36][37][38][39][40]. In E. coli HU 88 proteins are differentially expressed during the cell cycle. The αHU-βHU heterodimer is 89 prevalent in stationary phase, while during exponential growth HU is predominantly present 90 a...

No abstract

Fluorescence microscopy is a valuable tool to study a broad variety of bacterial cell components and dynamics thereof. For Clostridioides difficile, the fluorescent proteins CFP opt , mCherry Opt and phiLOV2.1, and the self-labelling tags SNAP Cd and HaloTag, hereafter collectively referred as fluorescent systems, have been described to explore different cellular pathways. In this study, we sought to characterize previously used fluorescent systems in C. difficile cells. We performed single cell analyses using fluorescence microscopy of exponentially growing 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 show that the intrinsic fluorescence of C. difficile cells increases during growth, independent of sigB or spo0A. However, when C. difficile cells are exposed to environmental oxygen autofluorescence is enhanced. Cytosolic overexpression of the different fluorescent systems alone, using the same expression signals, showed heterogeneous expression of the fluorescent systems. High levels of mCherry Opt were toxic for C. difficile cells limiting the applicability of this fluorophore as a transcriptional reporter. When fused to HupA, a C. difficile histone-like protein, the fluorescent systems behaved similarly and did not affect the HupA overproduction phenotype. 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. Due to independence of molecular oxygen and fluorescent signal, SNAP Cd appears the most suitable candidate for live-cell imaging in C. difficile to date.

Objectives: Fluorescence microscopy is a valuable tool to study a broad variety of bacterial cell components and dynamics thereof. 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. In this study, we sought to characterize previously used fluorescent systems in C. difficile cells. Methods: We performed single cell analyses using fluorescence microscopy of exponentially growing 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. Results: We show that the intrinsic fluorescence of C. difficile cells increases during growth, independent from sigB or spo0A. However, when C. difficile cells are exposed to environmental oxygen autofluorescence is enhanced. Cytosolic overexpression of the different fluorescent systems alone, using the same expression signals, showed heterogeneous expression of the fluorescent systems. High levels of mCherryOpt were toxic for C. difficile cells limiting the applicability of this fluorophore as a transcriptional reporter. When fused to HupA, C. difficile histone-like protein, the fluorescent systems behaved similarly and did not affect the HupA overproduction phenotype. Conclusions: 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. Due to independence of molecular oxygen and fluorescent signal, SNAPCd appears the most suitable candidate for live-cell imaging in C. difficile to date.