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

FtsZ does not initiate membrane constriction at the onset of division.

The source of constriction required for division of a bacterial cell remains enigmatic. FtsZ is widely believed to be a key player, because in vitro experiments indicate that it can deform liposomes when membrane tethered. However in vivo evidence for such a role has remained elusive as it has been challenging to distinguish the contribution of FtsZ from that of peptidoglycan-ingrowth. To differentiate between these two possibilities we studied the early stages of division in Escherichia coli, when FtsZ is present at the division site but peptidoglycan synthesizing enzymes such as FtsI and FtsN are not. Our approach was to use correlative cryo-fluorescence and cryo-electron microscopy (cryo-CLEM) to monitor the localization of fluorescently labeled FtsZ, FtsI or FtsN correlated with the septal ultra-structural geometry in the same cell. We noted that the presence of FtsZ at the division septum is not sufficient to deform membranes. This observation suggests that, although FtsZ can provide a constrictive force, the force is not substantial at the onset of division. Conversely, the presence of FtsN always correlated with membrane invagination, indicating that allosteric activation of peptidoglycan ingrowth is the trigger for constriction of the cell envelope during cell division in E. coli

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

Growth and characterization of ZnTe films grown on GaAs, InAs, GaSb, and ZnTe

We report the successful growth of ZnTe on nearly lattice-matched III-V buffer layers of InAs (0.75%), GaSb (0.15%), and on GaAs and ZnTe by molecular beam epitaxy. In situ reflection high-energy electron diffraction measurements showed the characteristic streak patterns indicative of two-dimensional growth. Photoluminescence measurements on these films show strong and sharp features near the band edge with no detectable luminescence at longer wavelengths. The integrated photoluminescence intensity from the ZnTe layers increased with better lattice match to the buffer layer. The ZnTe epilayers grown on high-purity ZnTe substrates exhibited stronger luminescence than the substrates. We observe narrow luminescence linewidths (full width at half maximum ~ 1–2 Å) indicative of uniform high quality growth. Secondary-ion mass spectroscopy and electron microprobe measurements, however, reveal substantial outdiffusion of Ga and In for growths on the III-V buffer layers

Detecting Gold Biomineralization by Delftia acidovorans Biofilms on a Quartz Crystal Microbalance

© 2019 American Chemical Society. The extensive use of gold in sensing, diagnostics, and electronics has led to major concerns in solid waste management since gold and other heavy metals are nonbiodegradable and can easily accumulate in the environment. Moreover, gold ions are extremely reactive and potentially harmful for humans. Thus, there is an urgent need to develop reliable methodologies to detect and possibly neutralize ionic gold in aqueous solutions and industrial wastes. In this work, by using complementary measurement techniques such as quartz crystal microbalance (QCM), atomic force microscopy, crystal violet staining, and optical microscopy, we investigate a promising biologically induced gold biomineralization process accomplished by biofilms of bacterium Delftia acidovorans. When stressed by Au3+ ions, D. acidovorans is able to neutralize toxic soluble gold by excreting a nonribosomal peptide, which forms extracellular gold nanonuggets via complexation with metal ions. Specifically, QCM, a surface-sensitive transducer, is employed to quantify the production of these gold complexes directly on the D. acidovorans biofilm in real time. Detailed kinetics obtained by QCM captures the condition for maximized biomineralization yield and offers new insights underlying the biomineralization process. To the best of our knowledge, this is the first study providing an extensive characterization of the gold biomineralization process by a model bacterial biofilm. We also demonstrate QCM as a cheap, user-friendly sensing platform and alternative to standard analytical techniques for studies requiring high-resolution quantitative details, which offers promising opportunities in heavy-metal sensing, gold recovery, and industrial waste treatment

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.</jats:p

Cell shape independent FtsZ dynamics in synthetically remodeled cells

The FtsZ protein is a key regulator of bacterial cell division. It has been implicated in acting as a scaffolding protein for other division proteins, being a force generator during constriction, and more recently, as an active regulator of septal cell wall production. During an early stage of the division cycle, FtsZ assembles into a heterogeneous structure coined the “Z-ring” due to its resemblance to a ring confined by the midcell geometry. While in vitro experiments on supported lipid bilayers have shown that purified FtsZ can self-organize into a swirling ring roughly the diameter of a bacterial cell, it is not known how, and if, membrane curvature affects FtsZ assembly and dynamics in vivo . To establish a framework for examining geometrical influences on proper Z-ring assembly and dynamics, we sculptured Escherichia coli cells into unnatural shapes, such as squares and hearts, 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”, and in giant cells up to 50 times their normal volume. Quantification of super-resolution STimulated Emission Depletion (STED) nanoscopy data showed that FtsZ densities in sculptured cells maintained the same dimensions as their wild-type counterparts. Additionally, time-resolved fluorescence measurements revealed that FtsZ dynamics were generally conserved in a wide range of cell shapes. Based on our results, we conclude that the underlying membrane environment is not a deciding factor for FtsZ filament maintenance and treadmilling in vivo

The bacterial DNA binding protein matp involved in linking the nucleoid terminal domain to the divisome at midcell interacts with lipid membranes

© 2019 Monterroso et al. Division ring formation at midcell is controlled by various mechanisms in Escherichia coli, one of them being the linkage between the chromosomal Ter macrodomain and the Z-ring mediated by MatP, a DNA binding protein that organizes this macrodomain and contributes to the prevention of premature chromosome segregation. Here we show that, during cell division, just before splitting the daughter cells, MatP seems to localize close to the cytoplasmic membrane, suggesting that this protein might interact with lipids. To test this hypothesis, we investigated MatP interaction with lipids in vitro. We found that, when encapsulated inside vesicles and microdroplets generated by microfluidics, MatP accumulates at phospholipid bilayers and monolayers matching the lipid composition in the E. coli inner membrane. MatP binding to lipids was independently confirmed using lipid-coated microbeads and biolayer interferometry assays, which suggested that the recognition is mainly hydrophobic. Interaction of MatP with the lipid membranes also occurs in the presence of the DNA sequences specifically targeted by the protein, but there is no evidence of ternary membrane/protein/DNA complexes. We propose that the association of MatP with lipids may modulate its spatiotemporal localization and its recognition of other ligands. IMPORTANCE The division of an E. coli cell into two daughter cells with equal genomic information and similar size requires duplication and segregation of the chromosome and subsequent scission of the envelope by a protein ring, the Z-ring. MatP is a DNA binding protein that contributes both to the positioning of the Z-ring at midcell and the temporal control of nucleoid segregation. Our integrated in vivo and in vitro analysis provides evidence that MatP can interact with lipid membranes reproducing the phospholipid mixture in the E. coli inner membrane, without concomitant recruitment of the short DNA sequences specifically targeted by MatP. This observation strongly suggests that the membrane may play a role in the regulation of the function and localization of MatP, which could be relevant for the coordination of the two fundamental processes in which this protein participates, nucleoid segregation and cell division