Cell constriction requires processive septal peptidoglycan synthase movement independent of FtsZ treadmilling in Staphylococcus aureus.

Bacterial cell division requires recruitment of peptidoglycan (PG) synthases to the division site by the tubulin homologue, FtsZ. Septal PG synthases promote septum growth. FtsZ treadmilling is proposed to drive the processive movement of septal PG synthases and septal constriction in some bacteria; however, the precise mechanisms spatio-temporally regulating PG synthase movement and activity and FtsZ treadmilling are poorly understood. Here using single-molecule imaging of division proteins in the Gram-positive pathogen Staphylococcus aureus, we showed that the septal PG synthase complex FtsW/PBP1 and its putative activator protein, DivIB, move with similar velocity around the division site. Impairing FtsZ treadmilling did not affect FtsW or DivIB velocities or septum constriction rates. Contrarily, PG synthesis inhibition decelerated or stopped directional movement of FtsW and DivIB, and septum constriction. Our findings suggest that a single population of processively moving FtsW/PBP1 associated with DivIB drives cell constriction independently of FtsZ treadmilling in S. aureus.

The role of GpsB in Staphylococcus aureus cell morphogenesis.

For decades, cells of the Gram-positive bacterial pathogen Staphylococcus aureus were thought to lack a dedicated elongation machinery. However, S. aureus cells were recently shown to elongate before division, in a process that requires a shape elongation division and sporulation (SEDS)/penicillin-binding protein (PBP) pair for peptidoglycan synthesis, consisting of the glycosyltransferase RodA and the transpeptidase PBP3. In ovococci and rod-shaped bacteria, the elongation machinery, or elongasome, is composed of various proteins besides a dedicated SEDS/PBP pair. To identify proteins required for S. aureus elongation, we screened the Nebraska Transposon Mutant Library, which contains transposon mutants in virtually all non-essential staphylococcal genes, for mutants with modified cell shape. We confirmed the roles of RodA/PBP3 in S. aureus elongation and identified GpsB, SsaA, and RodZ as additional proteins involved in this process. The gpsB mutant showed the strongest phenotype, mediated by the partial delocalization from the division septum of PBP2 and PBP4, two penicillin-binding proteins that synthesize and cross-link peptidoglycan. Increased levels of these PBPs at the cell periphery versus the septum result in higher levels of peptidoglycan insertion/crosslinking throughout the entire cell, possibly overriding the RodA/PBP3-mediated peptidoglycan synthesis at the outer edge of the septum and/or increasing stiffness of the peripheral wall, impairing elongation. Consequently, in the absence of GpsB, S. aureus cells become more spherical. We propose that GpsB has a role in the spatio-temporal regulation of PBP2 and PBP4 at the septum versus cell periphery, contributing to the maintenance of the correct cell morphology in S. aureus. IMPORTANCE: Staphylococcus aureus is a Gram-positive clinical pathogen, which is currently the second cause of death by antibiotic-resistant infections worldwide. For decades, S. aureus cells were thought to be spherical and lack the ability to undergo elongation. However, super-resolution microscopy techniques allowed us to observe the minor morphological changes that occur during the cell cycle of this pathogen, including cell elongation. S. aureus elongation is not required for normal growth in laboratory conditions. However, it seems to be essential in the context of some infections, such as osteomyelitis, during which S. aureus cells apparently elongate to invade small channels in the bones. In this work, we uncovered new determinants required for S. aureus cell elongation. In particular, we show that GpsB has an important role in the spatio-temporal regulation of PBP2 and PBP4, two proteins involved in peptidoglycan synthesis, contributing to the maintenance of the correct cell morphology in S. aureus.

A CRISPRi-based genetic resource to study essential Staphylococcus aureus genes.

IMPORTANCE: Staphylococcus aureus is an important clinical pathogen that causes a high number of antibiotic-resistant infections. The study of S. aureus biology, and particularly of the function of essential proteins, is of particular importance to develop new approaches to combat this pathogen. We have optimized a clustered regularly interspaced short palindromic repeat interference (CRISPRi) system that allows efficient targeting of essential S. aureus genes. Furthermore, we have used that system to construct a library comprising 261 strains, which allows the depletion of essential proteins encoded by 200 genes/operons. This library, which we have named Lisbon CRISPRi Mutant Library, should facilitate the study of S. aureus pathogenesis and biology.

Cell division protein FtsK coordinates bacterial chromosome segregation and daughter cell separation in Staphylococcus aureus.

Unregulated cell cycle progression may have lethal consequences and therefore, bacteria have various mechanisms in place for the precise spatiotemporal control of cell cycle events. We have uncovered a new link between chromosome replication/segregation and splitting of the division septum. We show that the DNA translocase domain-containing divisome protein FtsK regulates cellular levels of a peptidoglycan hydrolase Sle1, which is involved in cell separation in the bacterial pathogen Staphylococcus aureus. FtsK interacts with a chaperone (trigger factor, TF) and establishes a FtsK-dependent TF concentration gradient that is higher in the septal region. Trigger factor binds Sle1 and promotes its preferential export at the septal region, while also preventing Sle1 degradation by the ClpXP proteolytic machinery. Upon conditions that lead to paused septum synthesis, such as DNA damage or impaired DNA replication/segregation, TF gradient is dissipated and Sle1 levels are reduced, thus halting premature septum splitting.

Correlating Optical Reflectance with the Topology of Aluminum Nanocluster Layers Growing on Partially Conjugated Diblock Copolymer Templates.

Large-scale fabrication of metal cluster layers for usage in sensor applications and photovoltaics is a huge challenge. Physical vapor deposition offers large-scale fabrication of metal cluster layers on templates and polymer surfaces. In the case of aluminum (Al), only little is known about the formation and interaction of Al clusters during sputter deposition. Complex polymer surface morphologies can tailor the deposited Al cluster layer. Here, a poly(methyl methacrylate)-block-poly(3-hexylthiophen-2,5-diyl) (PMMA-b-P3HT) diblock copolymer template is used to investigate the nanostructure formation of Al cluster layers on the different polymer domains and to compare it with the respective homopolymers PMMA and P3HT. The optical properties relevant for sensor applications are monitored with ultraviolet-visible (UV-vis) measurements during the sputter deposition. The formation of Al clusters is followed in situ with grazing-incidence small-angle X-ray scattering (GISAXS), and the chemical interaction is revealed by X-ray photoelectron spectroscopy (XPS). Furthermore, atomic force microscopy (AFM) and field emission scanning electron microscopy (FESEM) yield topographical information about selective wetting of Al on the P3HT domains and embedding in the PMMA domains in the early stages, followed by four distinct growth stages describing the Al nanostructure formation.

Revealing the growth of copper on polystyrene-block-poly(ethylene oxide) diblock copolymer thin films with in situ GISAXS.

Copper (Cu) as an excellent electrical conductor and the amphiphilic diblock copolymer polystyrene-block-poly(ethylene oxide) (PS-b-PEO) as a polymer electrolyte and ionic conductor can be combined with an active material in composite electrodes for polymer lithium-ion batteries (LIBs). As interfaces are a key issue in LIBs, sputter deposition of Cu contacts on PS-b-PEO thin films with high PEO fraction is investigated with in situ grazing-incidence small-angle X-ray scattering (GISAXS) to follow the formation of the Cu layer in real-time. We observe a hierarchical morphology of Cu clusters building larger Cu agglomerates. Two characteristic distances corresponding to the PS-b-PEO microphase separation and the Cu clusters are determined. A selective agglomeration of Cu clusters on the PS domains explains the origin of the persisting hierarchical morphology of the Cu layer even after a complete surface coverage is reached. The spheroidal shape of the Cu clusters growing within the first few nanometers of sputter deposition causes a highly porous Cu-polymer interface. Four growth stages are distinguished corresponding to different kinetics of the cluster growth of Cu on PS-b-PEO thin films: (I) nucleation, (II) diffusion-driven growth, (III) adsorption-driven growth, and (IV) grain growth of Cu clusters. Percolation is reached at an effective Cu layer thickness of 5.75 nm.

Characterization and Quantification of Depletion and Accumulation Layers in Solid-State Li+ -Conducting Electrolytes Using In Situ Spectroscopic Ellipsometry.

The future of mobility depends on the development of next-generation battery technologies, such as all-solid-state batteries. As the ionic conductivity of solid Li+ -conductors can, in some cases, approach that of liquid electrolytes, a significant remaining barrier faced by solid-state electrolytes (SSEs) is the interface formed at the anode and cathode materials, with chemical instability and physical resistances arising. The physical properties of space charge layers (SCLs), a widely discussed phenomenon in SSEs, are still unclear. In this work, spectroscopic ellipsometry is used to characterize the accumulation and depletion layers. An optical model is developed to quantify their thicknesses and corresponding concentration changes. It is shown that the Li+ -depleted layer (≈190 nm at 1 V) is thinner than the accumulation layer (≈320 nm at 1 V) in a glassy lithium-ion-conducting glass ceramic electrolyte (a trademark of Ohara Corporation). The in situ approach combining electrochemistry and optics resolves the ambiguities around SCL formation. It opens up a wide field of optical measurements on SSEs, allowing various experimental studies in the future.

Tailoring the Optical Properties of Sputter-Deposited Gold Nanostructures on Nanostructured Titanium Dioxide Templates Based on In Situ Grazing-Incidence Small-Angle X-ray Scattering Determined Growth Laws.

Gold/titanium dioxide (Au/TiO2) nanohybrid materials have been widely applied in various fields because of their outstanding optical and photocatalytic performance. By state-of-the-art polymer templating, it is possible to make uniform nanostructured TiO2 layers with potentially large-scale processing methods. We use customized polymer templating to achieve TiO2 nanostructures with different morphologies. Au/TiO2 hybrid thin films are fabricated by sputter deposition. An in-depth understanding of the Au morphology on the TiO2 templates is achieved with in situ grazing-incidence small-angle X-ray scattering (GISAXS) during the sputter deposition. The resulting Au nanostructure is largely influenced by the TiO2 template morphology. Based on the detailed understanding of the Au growth process, characteristic distances can be selected to achieve tailored Au nanostructures at different Au loadings. For selected sputter-deposited Au/TiO2 hybrid thin films, the optical response with a tailored localized surface plasmon resonance is demonstrated.

Characterization of an active ingredient made of nanoscale iron(oxyhydr)oxide for the treatment of hyperphosphatemia.

Kidney disease is one of the main non-communicable diseases. Every year millions of people worldwide die from kidney dysfunction. One cause is disturbances in the mineral metabolism, such as abnormally high phosphate concentrations in the blood, medically referred to as hyperphosphatemia. A new active ingredient based on nanoscale iron(oxyhydr)oxide with particle sizes below 3 nm surrounded by an organic coating has been developed for a more effective treatment. The examination of the structural properties of these particles within this study promises to gain further insights into this improved effectiveness. More than half of the active ingredient consists of organic substances, the rest is mostly iron(oxyhydr)oxide. Analyzes by transmission electron microscopy (TEM), small-angle X-ray scattering (SAXS), and dynamic light scattering (DLS) show that the organic molecules act as stabilizers and lead to ultrasmall iron(oxyhydr)oxide cores with a size of 1.0-2.8 nm. The nanoparticles coated with the organic molecules have an average size of 11.7 nm. At 4.2 K, the nanoparticles display a magnetic hyperfine field of 45.5 T in the Mössbauer spectrum, which is unusually low for iron(oxyhydr)oxide. The material is also not ferrimagnetic. Combining these results and taking into account the composition of the nanoparticles, we identify low crystalline ferrihydrite as the most likely phase in the iron(oxyhydr)oxide nuclei. At the same time, we want to emphasize that a final identification of the crystal structure in iron(oxyhydr)oxides can be impeded by ultrasmall particle sizes. In summary, by a combinatorial characterization, we are able to observe extraordinary properties of the ultrasmall nanomaterial, which is the basis for the investigation of the high phosphate-binding efficacy of this active ingredient.

Real-time insight into nanostructure evolution during the rapid formation of ultra-thin gold layers on polymers.

Ultra-thin metal layers on polymer thin films attract tremendous research interest for advanced flexible optoelectronic applications, including organic photovoltaics, light emitting diodes and sensors. To realize the large-scale production of such metal-polymer hybrid materials, high rate sputter deposition is of particular interest. Here, we witness the birth of a metal-polymer hybrid material by quantifying in situ with unprecedented time-resolution of 0.5 ms the temporal evolution of interfacial morphology during the rapid formation of ultra-thin gold layers on thin polystyrene films. We monitor average non-equilibrium cluster geometries, transient interface morphologies and the effective near-surface gold diffusion. At 1 s sputter deposition, the polymer matrix has already been enriched with 1% gold and an intermixing layer has formed with a depth of over 3.5 nm. Furthermore, we experimentally observe unexpected changes in aspect ratios of ultra-small gold clusters growing in the vicinity of polymer chains. For the first time, this approach enables four-dimensional insights at atomic scales during the gold growth under non-equilibrium conditions.

Cyclic di-GMP signaling controlling the free-living lifestyle of alpha-proteobacterial rhizobia.

Cyclic-di-GMP (c-di-GMP) is a ubiquitous bacterial second messenger which has been associated with a motile to sessile lifestyle switch in many bacteria. Here, we review recent insights into c-di-GMP regulated processes related to environmental adaptations in alphaproteobacterial rhizobia, which are diazotrophic bacteria capable of fixing nitrogen in symbiosis with their leguminous host plants. The review centers on Sinorhizobium meliloti, which in the recent years was intensively studied for its c-di-GMP regulatory network.

In situ Grazing-Incidence Small-Angle X-ray Scattering Observation of Gold Sputter Deposition on a PbS Quantum Dot Solid.

For PbS quantum dot (QD)-based optoelectronic devices, gold is the most frequently used electrode material. In most device architectures, gold is in direct contact with the QD solid. To better understand the formation of the interface between gold and a close-packed QD layer at an early stage, in situ grazing-incidence small-angle X-ray scattering is used to observe the gold sputter deposition on a 1,2-ethanedithiol (EDT)-treated PbS QD solid. In the kinetics of gold layer growth, the forming and merging of small gold clusters (radius less than 1.6 nm) are observed at the early stages. The thereby formed medium gold clusters (radius between 1.9-2.4 nm) are influenced by the QDs' templating effect. Furthermore, simulations suggest that the medium gold clusters grow preferably along the QDs' boundaries rather than as a top coating of the QDs. When the thickness of the sputtered gold layer reaches 6.25 nm, larger gold clusters with a radius of 5.3 nm form. Simultaneously, a percolation layer with a thickness of 2.5 nm is established underneath the gold clusters. This fundamental understanding of the QD-gold interface formation will help to control the implementation of sputtered gold electrodes on close-packed QD solids in device manufacturing processes.

Tol-Pal System and Rgs Proteins Interact to Promote Unipolar Growth and Cell Division in Sinorhizobium meliloti.

Sinorhizobium meliloti is an alphaproteobacterium belonging to the Rhizobiales Bacteria from this order elongate their cell wall at the new cell pole, generated by cell division. Screening for protein interaction partners of the previously characterized polar growth factors RgsP and RgsM, we identified the inner membrane components of the Tol-Pal system (TolQ and TolR) and novel Rgs (rhizobial growth and septation) proteins with unknown functions. TolQ, Pal, and all Rgs proteins, except for RgsE, were indispensable for S. meliloti cell growth. Six of the Rgs proteins, TolQ, and Pal localized to the growing cell pole in the cell elongation phase and to the septum in predivisional cells, and three Rgs proteins localized to the growing cell pole only. The putative FtsN-like protein RgsS contains a conserved SPOR domain and is indispensable at the early stages of cell division. The components of the Tol-Pal system were required at the late stages of cell division. RgsE, a homolog of the Agrobacterium tumefaciens growth pole ring protein GPR, has an important role in maintaining the normal growth rate and rod cell shape. RgsD is a periplasmic protein with the ability to bind peptidoglycan. Analysis of the phylogenetic distribution of the Rgs proteins showed that they are conserved in Rhizobiales and mostly absent from other alphaproteobacterial orders, suggesting a conserved role of these proteins in polar growth.IMPORTANCE Bacterial cell proliferation involves cell growth and septum formation followed by cell division. For cell growth, bacteria have evolved different complex mechanisms. The most prevalent growth mode of rod-shaped bacteria is cell elongation by incorporating new peptidoglycans in a dispersed manner along the sidewall. A small share of rod-shaped bacteria, including the alphaproteobacterial Rhizobiales, grow unipolarly. Here, we identified and initially characterized a set of Rgs (rhizobial growth and septation) proteins, which are involved in cell division and unipolar growth of Sinorhizobium meliloti and highly conserved in Rhizobiales Our data expand the knowledge of components of the polarly localized machinery driving cell wall growth and suggest a complex of Rgs proteins with components of the divisome, differing in composition between the polar cell elongation zone and the septum.

The Dissociation Rate of Acetylacetonate Ligands Governs the Size of Ferrimagnetic Zinc Ferrite Nanocubes.

Magnetic nanoparticles are critical to a broad range of applications from medical diagnostics and therapeutics to biotechnological processes and single-molecule manipulation. To advance these applications, facile and robust routes to synthesize highly magnetic nanoparticles over a wide size range are needed. Here, we demonstrate that changing the degassing temperature of thermal decomposition of metal acetylacetonate precursors from 90 to 25 °C tunes the size of ferrimagnetic ZnxFe3-xO4 nanocubes from 25 to 100 nm, respectively. We show that degassing at 90 °C nearly entirely removes acetylacetone ligands from the reaction, which results in an early formation of monomers and a reaction-controlled growth following LaMer's model toward small nanocubes. In contrast, degassing at 25 °C only partially dissociates acetylacetone ligands from the metal center and triggers a delayed formation of monomers, which leads to intermediate assembled structures made of tiny irregular crystallites and an eventual formation of large nanocubes via a diffusion-controlled growth mechanism. Using complementary techniques, we determine the substitution fraction x of Zn2+ to be in the range of 0.35-0.37. Our method reduces the complexity of the thermal decomposition method by narrowing the synthesis parameter space to a single physical parameter and enables fabrication of highly magnetic and uniform zinc ferrite nanocubes over a broad size range. The resulting particles are promising for a range of applications from magnetic fluid hyperthermia to actuation of macromolecules.

A Bifunctional UDP-Sugar 4-Epimerase Supports Biosynthesis of Multiple Cell Surface Polysaccharides in Sinorhizobium meliloti.

Sinorhizobium meliloti produces multiple extracellular glycans, including among others, lipopolysaccharides (LPS), and the exopolysaccharides (EPS) succinoglycan (SG) and galactoglucan (GG). These polysaccharides serve cell protective roles. Furthermore, SG and GG promote the interaction of S. meliloti with its host Medicago sativa in root nodule symbiosis. ExoB has been suggested to be the sole enzyme catalyzing synthesis of UDP-galactose in S. meliloti (A. M. Buendia, B. Enenkel, R. Köplin, K. Niehaus, et al. Mol Microbiol 5:1519-1530, 1991, https://doi.org/10.1111/j.1365-2958.1991.tb00799.x). Accordingly, exoB mutants were previously found to be affected in the synthesis of the galactose-containing glycans LPS, SG, and GG and consequently, in symbiosis. Here, we report that the S. meliloti Rm2011 uxs1-uxe-apsS-apsH1-apsE-apsH2 (SMb20458-63) gene cluster directs biosynthesis of an arabinose-containing polysaccharide (APS), which contributes to biofilm formation, and is solely or mainly composed of arabinose. Uxe has previously been identified as UDP-xylose 4-epimerase. Collectively, our data from mutational and overexpression analyses of the APS biosynthesis genes and in vitro enzymatic assays indicate that Uxe functions as UDP-xylose 4- and UDP-glucose 4-epimerase catalyzing UDP-xylose/UDP-arabinose and UDP-glucose/UDP-galactose interconversions, respectively. Overexpression of uxe suppressed the phenotypes of an exoB mutant, evidencing that Uxe can functionally replace ExoB. We suggest that under conditions stimulating expression of the APS biosynthesis operon, Uxe contributes to the synthesis of multiple glycans and thereby to cell protection, biofilm formation, and symbiosis. Furthermore, we show that the C2H2 zinc finger transcriptional regulator MucR counteracts the previously reported CuxR-c-di-GMP-mediated activation of the APS biosynthesis operon. This integrates the c-di-GMP-dependent control of APS production into the opposing regulation of EPS biosynthesis and swimming motility in S. melilotiIMPORTANCE Bacterial extracellular polysaccharides serve important cell protective, structural, and signaling roles. They have particularly attracted attention as adhesives and matrix components promoting biofilm formation, which significantly contributes to resistance against antibiotics. In the root nodule symbiosis between rhizobia and leguminous plants, extracellular polysaccharides have a signaling function. UDP-sugar 4-epimerases are important enzymes in the synthesis of the activated sugar substrates, which are frequently shared between multiple polysaccharide biosynthesis pathways. Thus, these enzymes are potential targets to interfere with these pathways. Our finding of a bifunctional UDP-sugar 4-epimerase in Sinorhizobium meliloti generally advances the knowledge of substrate promiscuity of such enzymes and specifically of the biosynthesis of extracellular polysaccharides involved in biofilm formation and symbiosis in this alphaproteobacterium.

Seven-transmembrane receptor protein RgsP and cell wall-binding protein RgsM promote unipolar growth in Rhizobiales.

Members of the Rhizobiales (class of α-proteobacteria) display zonal peptidoglycan cell wall growth at one cell pole, contrasting with the dispersed mode of cell wall growth along the sidewalls of many other rod-shaped bacteria. Here we show that the seven-transmembrane receptor (7TMR) protein RgsP (SMc00074), together with the putative membrane-anchored peptidoglycan metallopeptidase RgsM (SMc02432), have key roles in unipolar peptidoglycan formation during growth and at mid-cell during cell division in Sinorhizobium meliloti. RgsP is composed of a periplasmic globular 7TMR-DISMED2 domain, a membrane-spanning region, and cytoplasmic PAS, GGDEF and EAL domains. The EAL domain confers phosphodiesterase activity towards the second messenger cyclic di-GMP, a key regulatory player in the transition between bacterial lifestyles. RgsP and RgsM localize to sites of zonal cell wall synthesis at the new cell pole and cell divison site, suggesting a role in cell wall biogenesis. The two proteins are essential for cell wall biogenesis and cell growth. Cells depleted of RgsP or RgsM had an altered muropeptide composition and RgsM binds to peptidoglycan. RgsP and RgsM orthologs are functional when interchanged between α-rhizobial species pointing to a conserved mechanism for cell wall biogenesis/remodeling within the Rhizobiales. Overall, our findings suggest that RgsP and RgsM contribute to the regulation of unipolar cell wall biogenesis in α-rhizobia.

AraC-like transcriptional activator CuxR binds c-di-GMP by a PilZ-like mechanism to regulate extracellular polysaccharide production.

Cyclic dimeric GMP (c-di-GMP) has emerged as a key regulatory player in the transition between planktonic and sedentary biofilm-associated bacterial lifestyles. It controls a multitude of processes including production of extracellular polysaccharides (EPSs). The PilZ domain, consisting of an N-terminal "RxxxR" motif and a ß-barrel domain, represents a prototype c-di-GMP receptor. We identified a class of c-di-GMP-responsive proteins, represented by the AraC-like transcription factor CuxR in plant symbiotic α-proteobacteria. In Sinorhizobium meliloti, CuxR stimulates transcription of an EPS biosynthesis gene cluster at elevated c-di-GMP levels. CuxR consists of a Cupin domain, a helical hairpin, and bipartite helix-turn-helix motif. Although unrelated in sequence, the mode of c-di-GMP binding to CuxR is highly reminiscent to that of PilZ domains. c-di-GMP interacts with a conserved N-terminal RxxxR motif and the Cupin domain, thereby promoting CuxR dimerization and DNA binding. We unravel structure and mechanism of a previously unrecognized c-di-GMP-responsive transcription factor and provide insights into the molecular evolution of c-di-GMP binding to proteins.

Cyclic Di-GMP Regulates Multiple Cellular Functions in the Symbiotic Alphaproteobacterium Sinorhizobium meliloti.

UNLABELLED: Sinorhizobium meliloti undergoes major lifestyle changes between planktonic states, biofilm formation, and symbiosis with leguminous plant hosts. In many bacteria, the second messenger 3',5'-cyclic di-GMP (c-di-GMP, or cdG) promotes a sessile lifestyle by regulating a plethora of processes involved in biofilm formation, including motility and biosynthesis of exopolysaccharides (EPS). Here, we systematically investigated the role of cdG in S. meliloti Rm2011 encoding 22 proteins putatively associated with cdG synthesis, degradation, or binding. Single mutations in 21 of these genes did not cause evident changes in biofilm formation, motility, or EPS biosynthesis. In contrast, manipulation of cdG levels by overproducing endogenous or heterologous diguanylate cyclases (DGCs) or phosphodiesterases (PDEs) affected these processes and accumulation of N-Acyl-homoserine lactones in the culture supernatant. Specifically, individual overexpression of the S. meliloti genes pleD, SMb20523, SMb20447, SMc01464, and SMc03178 encoding putative DGCs and of SMb21517 encoding a single-domain PDE protein had an impact and resulted in increased levels of cdG. Compared to the wild type, an S. meliloti strain that did not produce detectable levels of cdG (cdG(0)) was more sensitive to acid stress. However, it was symbiotically potent, unaffected in motility, and only slightly reduced in biofilm formation. The SMc01790-SMc01796 locus, homologous to the Agrobacterium tumefaciens uppABCDEF cluster governing biosynthesis of a unipolarly localized polysaccharide, was found to be required for cdG-stimulated biofilm formation, while the single-domain PilZ protein McrA was identified as a cdG receptor protein involved in regulation of motility. IMPORTANCE: We present the first systematic genome-wide investigation of the role of 3',5'-cyclic di-GMP (c-di-GMP, or cdG) in regulation of motility, biosynthesis of exopolysaccharides, biofilm formation, quorum sensing, and symbiosis in a symbiotic alpha-rhizobial species. Phenotypes of an S. meliloti strain unable to produce cdG (cdG(0)) demonstrated that this second messenger is not essential for root nodule symbiosis but may contribute to acid tolerance. Our data further suggest that enhanced levels of cdG promote sessility of S. meliloti and uncovered a single-domain PilZ protein as regulator of motility.

The conformational bases for the two functionalities of 2-cysteine peroxiredoxins as peroxidase and chaperone.

2-Cysteine peroxiredoxins (2-CysPrxs) are ubiquitous and highly abundant proteins that serve multiple functions as peroxidases, chaperones, and thiol oxidases and in redox-dependent cell signalling. The chloroplast protein plays a role in seedling development and protection of the photosynthetic apparatus. This study aimed to unequivocally link conformation and function. To this end, a set of non-tagged site-directed mutagenized At2-CysPrx variants was engineered, which mimicked the conformational states and their specific functions: hyperoxidized form (C54D), reduced form (C54S, C176S), oxidized form (C54DC176K), phosphorylated form (T92D), reduced ability for oligomerization by interfering with the dimer-dimer interface (F84R) and a C-terminally truncated form [ΔC (-20 aa)]. These variants were fully or partly fixed in their quaternary structure and function, respectively, and were analysed for their conformational state and peroxidase and chaperone activity, as well as for their sensitivity to hyperoxidation. The presence of a His6-tag strongly influenced the properties of the protein. The ΔC variant became insensitive to hyperoxidation, while T92D and F84R became more sensitive. The C54D variant revealed the highest chaperone activity. The highest peroxidase activity was observed for the F84R and ΔC variants. Efficient interaction with NADP-dependent thioredoxin reductase C depended on the presence of Cys residues and the C-terminal tail. The results suggest that the structural flexibility is important for the switch between peroxidase and chaperone function and that evolution has conserved the functional switch instead of maximizing a single function. These variants are ideal tools for future conformation-specific studies in vivo and in vitro.