Spo0J and SMC are required for normal chromosome segregation in Staphylococcus aureus

Bacterial chromosome segregation is an essential cellular process that is particularly elusive in spherical bacteria such as the opportunistic human pathogen Staphylococcus aureus. In this study, we examined the functional significance of a ParB homologue, Spo0J, in staphylococcal chromosome segregation and investigated the role of the structural maintenance of chromosomes (SMC) bacterial condensin in this process. We show that neither spo0J nor smc is essential in S. aureus; however, their absence causes abnormal chromosome segregation. We demonstrate that formation of complexes containing Spo0J and SMC is required for efficient S. aureus chromosome segregation and that SMC localization is dependent on Spo0J. Furthermore, we found that cell division and cell cycle progression are unaffected by the absence of spo0J or smc. Our results verify the role of Spo0J and SMC in ensuring accurate staphylococcal chromosome segregation and also imply functional redundancy or the involvement of additional mechanisms that might contribute to faithful chromosome inheritance

Cell shape-independent FtsZ dynamics in synthetically remodeled bacterial cells

FtsZ is the main regulator of bacterial cell division. It has been implicated in acting as a scaffolding protein for other division proteins, a force generator during constriction, and more recently, as an active regulator of septal cell wall production. FtsZ assembles into a heterogeneous structure coined the Z-ring due to its resemblance to a ring confined by the midcell geometry. Here, to establish a framework for examining geometrical influences on proper Z-ring assembly and dynamics, we sculpted Escherichia coli cells into unnatural shapes using division- and cell wall-specific inhibitors in a micro-fabrication scheme. This approach allowed us to examine FtsZ behavior in engineered Z-squares and Z-hearts. We use stimulated emission depletion (STED) nanoscopy to show that FtsZ clusters in sculpted cells maintain the same dimensions as their wild-type counterparts. Based on our results, we propose that the underlying membrane geometry is not a deciding factor for FtsZ cluster maintenance and dynamics in vivo

Application of nanotags and nanobodies for live cell single-molecule imaging of the Z-ring in Escherichia coli

Understanding where proteins are localized in a bacterial cell is essential for understanding their function and regulation. This is particularly important for proteins that are involved in cell division, which localize at the division septum and assemble into highly regulated complexes. Current knowledge of these complexes has been greatly facilitated by super-resolution imaging using fluorescent protein fusions. Herein, we demonstrate with FtsZ that single-molecule PALM images can be obtained in-vivo using a genetically fused nanotag (ALFA), and a corresponding nanobody fused to mEos3.2. The methodology presented is applicable to other bacterial proteins.journal articl

An OregonGreen488-labelled d-amino acid for visualizing peptidoglycan by super-resolution STED nanoscopy

Fluorescent d-amino acids (FDAAs) are molecular probes that are widely used for labelling the peptidoglycan layer of bacteria. When added to growing cells they are incorporated into the stem peptide by a transpeptidase reaction, allowing the timing and localization of peptidoglycan synthesis to be determined by fluorescence microscopy. Herein we describe the chemical synthesis of an OregonGreen488-labelled FDAA (OGDA). We also demonstrate that OGDA can be efficiently incorporated into the PG of Gram-positive and some Gram-negative bacteria, and imaged by super-resolution stimulated emission depletion (STED) nanoscopy at a resolution well below 100 nm

Nanoplasmonics for Real-Time and Label-Free Monitoring of Microbial Biofilm Formation

Microbial biofilms possess intrinsic resistance against conventional antibiotics and cleaning procedures; thus, a better understanding of their complex biological structures is crucial in both medical and industrial applications. Existing laboratory methodologies have focused on macroscopic and mostly indirect characterization of mechanical and microbiological properties of biofilms adhered on a given substrate. However, the kinetics underlying the biofilm formation is not well understood, while such information is critical to understanding how drugs and chemicals influence the biofilm formation. Herein, we report the use of localized surface plasmon resonance (LSPR) for real-time, label-free monitoring of E. coli biofilm assembly on a nanoplasmonic substrate consisting of gold mushroom-like structures. Our LSPR sensor is able to capture the signatures of biofilm formation in real-time by measuring the wavelength shift in the LSPR resonance peak with high temporal resolution. We employ this sensor feature to elucidate how biofilm formation is affected by different drugs, including conventional antibiotics (kanamycin and ampicillin) as well as rifapentine, a molecule preventing cell adhesion yet barely affecting bacterial viability and vitality. Due to its flexibility and simplicity, our LSPR based platform can be used on a wide variety of clinically relevant bacteria, thus representing a valuable tool in biofilm characterization and drug screening

Genetic requirements for uropathogenic E. coli proliferation in the bladder cell infection cycle

Uropathogenic Escherichia coli (UPEC) requires an adaptable physiology to survive the wide range of environments experienced in the host, including gut and urinary tract surfaces. To identify UPEC genes required during intracellular infection, we developed a transposon-directed insertion-site sequencing approach for cellular infection models and searched for genes in a library of ~20,000 UTI89 transposon-insertion mutants that are specifically required at the distinct stages of infection of cultured bladder epithelial cells. Some of the bacterial functional requirements apparent in host bladder cell growth overlapped with those for M9-glycerol, notably nutrient utilization, polysaccharide and macromolecule precursor biosynthesis, and cell envelope stress tolerance. Two genes implicated in the intracellular bladder cell infection stage were confirmed through independent gene deletion studies: neuC (sialic acid capsule biosynthesis) and hisF (histidine biosynthesis). Distinct sets of UPEC genes were also implicated in bacterial dispersal, where UPEC erupts from bladder cells in highly filamentous or motile forms upon exposure to human urine, and during recovery from infection in a rich medium. We confirm that the dedD gene linked to septal peptidoglycan remodeling is required during UPEC dispersal from human bladder cells and may help stabilize cell division or the cell wall during envelope stress created by host cells. Our findings support a view that the host intracellular environment and infection cycle are multi-nutrient limited and create stress that demands an array of biosynthetic, cell envelope integrity, and biofilm-related functions of UPEC

Nanoscale dynamics of peptidoglycan assembly during the cell cycle of Streptococcus pneumoniae

Dynamics of cell elongation and septation are key determinants of bacterial morphogenesis. These processes are intimately linked to peptidoglycan synthesis performed by macromolecular complexes called the elongasome and the divisome. In rod-shaped bacteria, cell elongation and septation, which are dissociated in time and space, have been well described. By contrast, in ovoid-shaped bacteria, the dynamics and relationships between these processes remain poorly understood because they are concomitant and confined to a nanometer-scale annular region at midcell. Here, we set up a metabolic peptidoglycan labeling approach using click chemistry to image peptidoglycan synthesis by single-molecule localization microscopy in the ovoid bacterium Streptococcus pneumoniae. Our nanoscale-resolution data reveal spatiotemporal features of peptidoglycan assembly and fate along the cell cycle and provide geometrical parameters that we used to construct a morphogenesis model of the ovoid cell. These analyses show that septal and peripheral peptidoglycan syntheses first occur within a single annular region that later separates in two concentric regions and that elongation persists after septation is completed. In addition, our data reveal that freshly synthesized peptidoglycan is remodeled all along the cell cycle. Altogether, our work provides evidence that septal peptidoglycan is synthesized from the beginning of the cell cycle and is constantly remodeled through cleavage and insertion of material at its periphery. The ovoid-cell morphogenesis would thus rely on the relative dynamics between peptidoglycan synthesis and cleavage rather than on the existence of two distinct successive phases of peripheral and septal synthesis

Restriction in the membrane diffusion over the dividing septum of Escherichia Coli cells measured by Flourescence Recovery After Photobleaching

This diploma thesis is a follow up work of a previous paper by Johan Strömqvist et. al. [1] In their article they presented a novel method on how to determine the Z-ring radius and the contraction in dividing Escherichia coli. This was done by fluorescent recovery after photobleaching (FRAP) measurements in the cytosol of E.coli cells transfected to express the Enhanced Green Fluorescent Protein (EGFP). The E.coli cell was irreversibly bleached on one side of an already visible invagination in the midcell, and then the fluorescence recovery was followed in time. Since the fluorescence recovery depends on the cross sectional area of the septum that information could be used to derive a mathematical expression to estimate the septal radius of the cell. In this thesis the goal was to, along with cytosolic recovery, examine the recovery after a bleaching pulse in the fluorescent labeled cell membrane. Furthermore it also set out to investigate how the cytosolic fluorescence recovery of EGFP was or could be correlated to the fluorescence recovery of dyes in the cell membranes of the E.coli

Fluorescence Studies of Cell Division in Escherichia coli

In Escherichia coli the cell division is carried out by a large dynamic protein complex called the divisome. The divisome assembles in a two-step manner starting with the localization of the eukaryotic tubulin homologue FtsZ to the midcell. Together with other early arriving proteins FtsZ form an intermediate structure called the Z-ring. After a considerable time lag the divisome maturates fully by recruiting several other late arriving proteins before it starts to constrict the cell envelope that ultimately will lead to cytokinesis and the formation of two identical daughter cells. Despite of being objectives of extensive study over the last decades, understanding of the exact molecular roles of many of the divisome proteins is still lacking and to date there is very limited knowledge of the disassembly process of the divisome. In this thesis I have used various fluorescence microscopy based methods to better characterize the role of FtsZ and other divisome proteins during the final stages of the cell division. I have shown that FtsZ disassembles from the divisome prior to inner membrane closure indicating that it is not the force generator during this final step of division that it is widely thought to be. I have also shown that the disassembly of the divisome is a multistep process in which the proteins that arrive in the second step of divisome assembly also remain at the division septum longer than those proteins that arrive in the first step. These findings add new important information regarding the cell division and together they provide a more complete picture of this event that ultimately may lead to more efficient identification of novel antibiotic targets.At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.</p