Assessing microbiome population dynamics using wild-type isogenic standardized hybrid (WISH)-tags.

Microbiomes feature recurrent compositional structures under given environmental conditions. However, these patterns may conceal diverse underlying population dynamics that require intrastrain resolution. Here we developed a genomic tagging system, termed wild-type isogenic standardized hybrid (WISH)-tags, that can be combined with quantitative polymerase chain reaction and next-generation sequencing for microbial strain enumeration. We experimentally validated the performance of 62 tags and showed that they can be differentiated with high precision. WISH-tags were introduced into model and non-model bacterial members of the mouse and plant microbiota. Intrastrain priority effects were tested using one species of isogenic barcoded bacteria in the murine gut and the Arabidopsis phyllosphere, both with and without microbiota context. We observed colonization resistance against late-arriving strains of Salmonella Typhimurium in the mouse gut, whereas the phyllosphere accommodated Sphingomonas latecomers in a manner proportional to their presence at the late inoculation timepoint. This demonstrates that WISH-tags are a resource for deciphering population dynamics underlying microbiome assembly across biological systems.

Regulation of Aerobic Succinate Transporter dctA of E. coli by cAMP-CRP, DcuS-DcuR, and EIIAGlc: Succinate as a Carbon Substrate and Signaling Molecule.

INTRODUCTION: C4-dicarboxylates (C4-DC) have emerged as significant growth substrates and signaling molecules for various Enterobacteriaceae during their colonization of mammalian hosts. Particularly noteworthy is the essential role of fumarate respiration during colonization of pathogenic bacteria. To investigate the regulation of aerobic C4-DC metabolism, the study explored the transcriptional control of the main aerobic C4-DC transporter, dctA, under different carbohydrate conditions. In addition, mutants related to carbon catabolite repression (CCR) and C4-DC regulation (DcuS-DcuR) were examined to better understand the regulatory integration of aerobic C4-DC metabolism into CCR. For initial insight into posttranslational regulation, the interaction between the aerobic C4-DC transporter DctA and EIIAGlc from the glucose-specific phosphotransferase system was investigated. METHODS: The expression of dctA was characterized in the presence of various carbohydrates and regulatory mutants affecting CCR. This was accomplished by fusing the dctA promoter (PdctA) to the lacZ reporter gene. Additionally, the interaction between DctA and EIIAGlc of the glucose-specific phosphotransferase system was examined in vivo using a bacterial two-hybrid system. RESULTS: The dctA promoter region contains a class I cAMP-CRP-binding site at position -81.5 and a DcuR-binding site at position -105.5. DcuR, the response regulator of the C4-DC-activated DcuS-DcuR two-component system, and cAMP-CRP stimulate dctA expression. The expression of dctA is subject to the influence of various carbohydrates via cAMP-CRP, which differently modulate cAMP levels. Here we show that EIIAGlc of the glucose-specific phosphotransferase system strongly interacts with DctA, potentially resulting in the exclusion of C4-DCs when preferred carbon substrates, such as sugars, are present. In contrast to the classical inducer exclusion known for lactose permease LacY, inhibition of C4-DC uptake into the cytoplasm affects only its role as a substrate, but not as an inducer since DcuS detects C4-DCs in the periplasmic space ("substrate exclusion"). The work shows an interplay between cAMP-CRP and the DcuS-DcuR regulatory system for the regulation of dctA at both transcriptional and posttranslational levels. CONCLUSION: The study highlights a hierarchical interplay between global (cAMP-CRP) and specific (DcuS-DcuR) regulation of dctA at the transcriptional and posttranslational levels. The integration of global and specific transcriptional regulation of dctA, along with the influence of EIIAGlc on DctA, fine-tunes C4-DC catabolism in response to the availability of other preferred carbon sources. It attributes DctA a central role in the control of aerobic C4-DC catabolism and suggests a new role to EIIAGlc on transporters (control of substrate uptake by substrate exclusion).

Fumarate, a central electron acceptor for Enterobacteriaceae beyond fumarate respiration and energy conservation.

C4-dicarboxylates (C4-DCs) such as fumarate, l-malate and l-aspartate are key substrates for Enterobacteria such as Escherichia coli or Salmonella typhimurium during anaerobic growth. In general, C4-DCs are oxidants during biosynthesis, e.g., of pyrimidine or heme, acceptors for redox balancing, a high-quality nitrogen source (l-aspartate) and electron acceptor for fumarate respiration. Fumarate reduction is required for efficient colonization of the murine intestine, even though the colon contains only small amounts of C4-DCs. However, fumarate can be produced endogenously by central metabolism, allowing autonomous production of an electron acceptor for biosynthesis and redox balancing. Bacteria possess a complex set of transporters for the uptake (DctA), antiport (DcuA, DcuB, TtdT) and excretion (DcuC) of C4-DCs. DctA and DcuB exert regulatory functions and link transport to metabolic control through interaction with regulatory proteins. The sensor kinase DcuS of the C4-DC two-component system DcuS-DcuR forms complexes with DctA (aerobic) or DcuB (anaerobic), representing the functional state of the sensor. Moreover, EIIAGlc from the glucose phospho-transferase system binds to DctA and presumably inhibits C4-DC uptake. Overall, the function of fumarate as an oxidant in biosynthesis and redox balancing explains the pivotal role of fumarate reductase for intestinal colonization, while the role of fumarate in energy conservation (fumarate respiration) is of minor importance.

C4-dicarboxylate metabolons: interaction of C4-dicarboxylate transporters of Escherichia coli with cytosolic enzymes.

Metabolons represent the structural organization of proteins for metabolic or regulatory pathways. Here, the interaction of fumarase FumB, aspartase AspA, and L-tartrate dehydratase TtdAB with the C4-dicarboxylate (C4-DC) transporters DcuA, DcuB, DcuC, and the L-tartrate transporter TtdT of Escherichia coli was tested by a bacterial two-hybrid (BACTH) assay in situ, or by co-chromatography using mSPINE (membrane Streptavidin protein interaction experiment). From the general C4-DC transporters, DcuB interacted with FumB and AspA, DcuA with AspA, whereas DcuC interacted with neither FumB nor AspA. Moreover, TtdT did not interact with TtdAB. The fumB-dcuB, the dcuA-aspA, and the ttdAB-ttdT genes encoding the respective proteins colocalize on the genome and each pair of genes forms cotranscripts, whereas the dcuC gene lies alone. The data suggest the formation of DcuB/FumB and DcuB/AspA metabolons for the uptake of L-malate, or L-aspartate, and their conversion to fumarate for fumarate respiration and excretion of the product succinate. The DcuA/AspA metabolon catalyzes uptake and conversion of L-aspartate to fumarate coupled to succinate excretion. The DcuA/AspA metabolon provides ammonia at the same time for nitrogen assimilation (ammonia shuttle). On the other hand, TtdT and TtdAB are not organized in a metabolon. Reasons for the formation (DcuA/AspA, DcuB/FumB, and DcuB/AspA) or nonformation (DcuC, TtdT, and TtdAB) of metabolons are discussed based on their metabolic roles.

C4-Dicarboxylates as Growth Substrates and Signaling Molecules for Commensal and Pathogenic Enteric Bacteria in Mammalian Intestine.

The C4-dicarboxylates (C4-DC) l-aspartate and l-malate have been identified as playing an important role in the colonization of mammalian intestine by enteric bacteria, such as Escherichia coli and Salmonella enterica serovar Typhimurium, and succinate as a signaling molecule for host-enteric bacterium interaction. Thus, endogenous and exogenous fumarate respiration and related functions are required for efficient initial growth of the bacteria. l-Aspartate represents a major substrate for fumarate respiration in the intestine and a high-quality substrate for nitrogen assimilation. During nitrogen assimilation, DcuA catalyzes an l-aspartate/fumarate antiport and serves as a nitrogen shuttle for the net uptake of ammonium only, whereas DcuB acts as a redox shuttle that catalyzes the l-malate/succinate antiport during fumarate respiration. The C4-DC two-component system DcuS-DcuR is active in the intestine and responds to intestinal C4-DC levels. Moreover, in macrophages and in mice, succinate is a signal that promotes virulence and survival of S. Typhimurium and pathogenic E. coli. On the other hand, intestinal succinate is an important signaling molecule for the host and activates response and protective programs. Therefore, C4-DCs play a major role in supporting colonization of enteric bacteria and as signaling molecules for the adaptation of host physiology.

Conversion of the Sensor Kinase DcuS to the Fumarate Sensitive State by Interaction of the Bifunctional Transporter DctA at the TM2/PASC-Linker Region.

The membrane-bound C4-dicarboxylate (C4DC) sensor kinase DcuS of Escherichia coli typically forms a protein complex with the C4DC transporter DctA. The DctA × DcuS complex is able to respond to C4DCs, whereas DcuS without DctA is in the permanent ON state. In DctA, the C-terminal helix 8b (H8b) serves as the site for interaction with DcuS. Here the interaction site in DcuS and the related structural and functional adaptation in DcuS were determined. The Linker connecting transmembrane helix 2 (TM2) and the cytosolic PASC (Per-ARNT-SIM) domain of DcuS, was identified as the major site for interaction with DctA-H8b by in vivo interaction studies. The Linker is known to convert the piston-type transmembrane signaling of TM2 to a tilting motion which relies on a resolution of the Linker-Linker' homodimer in the presence of C4DCs. Absence of DctA caused decreased cross-linking in the Linker, as identified by oxidative Cys-cross-linking. This response resembled structurally and functionally that of fumarate activation in the DctA × DcuS complex. Overall, formation of the DctA × DcuS complex is based on the interaction of the DcuS Linker with DctA H8b; the interaction is required to set DcuS in the C4DC-responsive state by stabilizing the linker-linker' homodimer in DcuS. This work identifies DctA as a structural co-regulator of DcuS sensor kinase.

C4-dicarboxylates and l-aspartate utilization by Escherichia coli K-12 in the mouse intestine: l-aspartate as a major substrate for fumarate respiration and as a nitrogen source.

C4-dicarboxylates, such as fumarate, l-malate and l-aspartate represent substrates for anaerobic growth of Escherichia coli by fumarate respiration. Here, we determined whether C4-dicarboxylate metabolism, as well as fumarate respiration, contribute to colonization of the mammalian intestinal tract. Metabolite profiling revealed that the murine small intestine contained high and low levels of l-aspartate and l-malate respectively, whereas fumarate was nearly absent. Under laboratory conditions, addition of C4-dicarboxylate at concentrations corresponding to the levels of the C4-dicarboxylates in the small intestine (2.6 mmol kg-1 dry weight) induced the dcuBp-lacZ reporter gene (67% of maximal) in a DcuS-DcuR-dependent manner. In addition to its role as a precursor for fumarate respiration, l-aspartate was able to supply all the nitrogen required for anaerobically growing E. coli. DcuS-DcuR-dependent genes were transcribed in the murine intestine, and mutants with defective anaerobic C4-dicarboxylate metabolism (dcuSR, frdA, dcuB, dcuA and aspA genes) were impaired for colonizing the murine gut. We conclude that l-aspartate plays an important role in providing fumarate for fumarate respiration and supplying nitrogen for E. coli in the mouse intestine.

L-Aspartate as a high-quality nitrogen source in Escherichia coli: Regulation of L-aspartase by the nitrogen regulatory system and interaction of L-aspartase with GlnB.

Escherichia coli uses the C4-dicarboxylate transporter DcuA for L-aspartate/fumarate antiport, which results in the exploitation of L-aspartate for fumarate respiration under anaerobic conditions and for nitrogen assimilation under aerobic and anaerobic conditions. L-Aspartate represents a high-quality nitrogen source for assimilation. Nitrogen assimilation from L-aspartate required DcuA, and aspartase AspA to release ammonia. Ammonia is able to provide by established pathways the complete set of intracellular precursors (ammonia, L-aspartate, L-glutamate, and L-glutamine) for synthesizing amino acids, nucleotides, and amino sugars. AspA was regulated by a central regulator of nitrogen metabolism, GlnB. GlnB interacted with AspA and stimulated its L-aspartate deaminase activity (NH3 -forming), but not the reverse amination reaction. GlnB stimulation required 2-oxoglutarate and ATP, or uridylylated GlnB-UMP, consistent with the activation of nitrogen assimilation under nitrogen limitation. Binding to AspA was lost in the GlnB(Y51F) mutant of the uridylylation site. AspA, therefore, represents a new type of GlnB target that binds GlnB (with ATP and 2-oxoglutarate), or GlnB-UMP (with or without effectors), and both situations stimulate AspA deamination activity. Thus, AspA represents the central enzyme for nitrogen assimilation from L-aspartate, and AspA is integrated into the nitrogen assimilation network by the regulator GlnB.

Transmembrane signaling and cytoplasmic signal conversion by dimeric transmembrane helix 2 and a linker domain of the DcuS sensor kinase.

Transmembrane (TM) signaling is a key process of membrane-bound sensor kinases. The C4-dicarboxylate (fumarate) responsive sensor kinase DcuS of Escherichia coli is anchored by TM helices TM1 and TM2 in the membrane. Signal transmission across the membrane relies on the piston-type movement of the periplasmic part of TM2. To define the role of TM2 in TM signaling, we use oxidative Cys cross-linking to demonstrate that TM2 extends over the full distance of the membrane and forms a stable TM homodimer in both the inactive and fumarate-activated state of DcuS. An S186xxxGxxxG194 motif is required for the stability and function of the TM2 homodimer. The TM2 helix further extends on the periplasmic side into the α6-helix of the sensory PASP domain and on the cytoplasmic side into the α1-helix of PASC. PASC has to transmit the signal to the C-terminal kinase domain. A helical linker on the cytoplasmic side connecting TM2 with PASC contains an LxxxLxxxL sequence. The dimeric state of the linker was relieved during fumarate activation of DcuS, indicating structural rearrangements in the linker. Thus, DcuS contains a long α-helical structure reaching from the sensory PASP (α6) domain across the membrane to α1(PASC). Taken together, the results suggest piston-type TM signaling by the TM2 homodimer from PASP across the full TM region, whereas the fumarate-destabilized linker dimer converts the signal on the cytoplasmic side for PASC and kinase regulation.

CyaC, a redox-regulated adenylate cyclase of Sinorhizobium meliloti with a quinone responsive diheme-B membrane anchor domain.

The nucleotide cyclase CyaC of Sinorhizobium meliloti is a member of class III adenylate cyclases (AC), a diverse group present in all forms of life. CyaC is membrane-integral by a hexahelical membrane domain (6TM) with the basic topology of mammalian ACs. The 6TM domain of CyaC contains a tetra-histidine signature that is universally present in the membrane anchors of bacterial diheme-B succinate-quinone oxidoreductases. Heterologous expression of cyaC imparted activity for cAMP formation from ATP to Escherichia coli, whereas guanylate cyclase activity was not detectable. Detergent solubilized and purified CyaC was a diheme-B protein and carried a binuclear iron-sulfur cluster. Single point mutations in the signature histidine residues caused loss of heme-B in the membrane and loss of AC activity. Heme-B of purified CyaC could be oxidized or reduced by ubiquinone analogs (Q0 or Q0 H2 ). The activity of CyaC in bacterial membranes responded to oxidation or reduction by Q0 and O2 , or NADH and Q0 H2 respectively. We conclude that CyaC-like membrane anchors of bacterial ACs can serve as the input site for chemical stimuli which are translated by the AC into an intracellular second messenger response.

The Influence of Carbosilane Nanosegregation on the Dynamics in 'de Vries-type' Liquid Crystals.

The mesogens QL32-6, QL33-6 and QL-34-6 contain 5-phenylpyrimidine cores and terminal nanosegregating carbosilane end groups of different lengths and are known to exhibit 'de Vries-type' properties of varying strength. We report a systematic study of the influence of the nanosegregating sublayer on the dynamics and rotational viscosities of the collective modes in the smectic A* (SmA*) and smectic C* (SmC*) phase using dielectric spectroscopy. It was found that the dynamics of the Goldstone mode corresponding to phase angle fluctuations are almost not affected while the relaxation time and rotational viscosity of the soft mode are influenced by the degree of nanosegregation. In other words, the nanosegregating sublayer does not influence the dynamics of ferroelectric switching in the SmC* phase, but is critical in inducing 'de Vries-type' properties.

DcuA of aerobically grown Escherichia coli serves as a nitrogen shuttle (L-aspartate/fumarate) for nitrogen uptake.

DcuA of Escherichia coli is known as an alternative C4 -dicarboxylate transporter for the main anaerobic C4 -dicarboxylate transporter DcuB. Since dcuA is expressed constitutively under aerobic and anaerobic conditions, DcuA was suggested to serve aerobically as a backup for the aerobic (DctA) transporter, or for the anabolic uptake of C4 -dicarboxylates. In this work, it is shown that DcuA is required for aerobic growth with L-aspartate as a nitrogen source, whereas for growth with L-aspartate as a carbon source, DctA was needed. Strains with DcuA catalyzed L-aspartate and C4 -dicarboxylate uptake (like DctA), or an L-aspartate/C4 -dicarboxylate antiport (unlike DctA). DcuA preferred L-aspartate to succinate in transport (KM = 43 and 844 µM, respectively), whereas DctA has higher affinity for C4 -dicarboxylates like succinate compared to L-aspartate. When L-aspartate was supplied as the sole nitrogen source together with glycerol as the carbon source, L-aspartate was taken up by the bacteria and fumarate (or L-malate) was excreted in equimolar amounts. Both reactions depended on DcuA. L-Aspartate was taken up in amounts required for nitrogen metabolism but not for carbon metabolism. Therefore, DcuA catalyzes an L-aspartate/C4 -dicarboxylate antiport serving as a nitrogen shuttle for nitrogen supply without net carbon supply.

Design of liquid crystals with 'de Vries-like' properties: structural variants of carbosilane-terminated 5-phenylpyrimidine mesogens.

Structural variants of the 'de Vries-like' mesogen 5-[4-(12,12,14,14,16,16-hexamethyl-12,14,16-trisilaheptadecyloxy)phenyl]-2-hexyloxypyrimidine (QL16-6), including two isomers with branched iso-tricarbosilane end-groups, were synthesized and their mesomorphic and 'de Vries-like' properties were characterized by polarized optical microscopy, differential scanning calorimetry, small angle and 2D X-ray scattering techniques. A comparative analysis of isomers with linear and branched tricarbosilane end-groups shows that they exhibit comparable mesomorphic and 'de Vries-like' properties. Furthermore, the difference in effective molecular length Leff between the linear and branched isomers in the SmA and SmC phases (ca. 4-5 Å), which was derived from 2D X-ray scattering experiments, suggests that the linear tricarbosilane end-group is hemispherical in shape on the time-average, as predicted by a DFT conformational analysis at the B3LYP/6-31G* level.

Electroclinic effect in a chiral carbosilane-terminated 5-phenylpyrimidine liquid crystal with 'de Vries-like' properties.

The chiral carbosilane-terminated liquid crystal 2-[(2S,3S)-2,3-difluorohexyloxy]-5-[4-(12,12,14,14,16,16-hexamethyl-12,14,16-trisilaheptadecyloxy)phenyl]pyrimidine () undergoes a smectic A*-smectic C* phase transition with a maximum layer contraction of only 0.2%. It exhibits an electroclinic effect (ECE) comparable to that reported for the 'de Vries-like' liquid crystal and shows no appreciable optical stripe defects due to horizontal chevron formation.

The Design and Investigation of Nanocomposites Containing Dimeric Nematogens and Liquid Crystal Gold Nanoparticles with Plasmonic Properties Showing a Nematic-Nematic Phase Transition (Nu-Nx/Ntb).

The construction of liquid crystal compositions consisting of the dimeric liquid crystal, CB_C9_CB (cyanobiphenyl dimer = 1'',9''-bis(4-cyanobiphenyl-4'-yl)nonane), and the range of nematic systems is explored. The materials include a laterally functionalized monomer, which was used to construct a phase diagram with CB_C9_CB, as well as one laterally linked dimer liquid crystal material and two liquid crystal gold nanoparticle (LC-Au-NPs) systems. For the Au-NP-LCs, the NP diameters were varied between ~3.3 nm and 10 nm. Stable mixtures that exhibit a nematic-nematic phase transition are reported and were investigated by POM (polarizing optical microscopy), DSC (differential scanning calorimetry) and X-ray diffraction studies.

Nematic molecular core flexibility and chiral induction.

Electroclinic measurements, in which an applied electric field E induces a rotation Δθ ([proportional]E) of the liquid crystal director about the electric field axis in a chiral environment, were performed on several configurationally achiral liquid crystals in the presence of an imposed helical director profile. This imposed twist establishes a chiral symmetry environment for the liquid crystal. It was observed that a conformationally racemic mesogen possessing a flexible phenyl benzoate core exhibits a measurable electroclinic response in the nematic phase. On the other hand, when the phenyl benzoate mesogen is mixed with a mesogen containing a rigid, conformationally achiral core (fluorenone), or with a racemic dopant with an axially chiral core that mimics a mesogen having rigid right- and left-handed conformations (2,2'-spirobiindan-1,1'-dione), the magnitudes of the electroclinic responses were found to decrease sharply, apparently going to zero when extrapolated to the pure 2,2'-spirobiindan-1,1'-dione or fluorenone limit. (Note that neither of these additives possesses a nematic phase.). The results suggest that the flexibility of the core and its ability to deracemize conformationally in order to compensate the elastic energy cost of the imposed twist is the primary mechanism behind the observed electroclinic response.

Chiral nematic organo-siloxane oligopodes based on an axially chiral binaphthalene core.

The synthesis of a novel class of organosiloxane oligopodes, based on an axially chiral binaphthalene core is described and their mesogenic properties are fully characterised.

Design, synthesis, and characterization of mesogenic amine-capped nematic gold nanoparticles with surface-enhanced plasmonic resonances.

The use of the liquid-crystalline state to control the assembly of large (>5 nm) gold nanoparticles (NPs) is of considerable interest because of the promise of novel metamaterial properties of such systems. Here we report on a new approach for the preparation of large nematic gold NPs using a bifunctional capping agent that enables control over the particle size and serves as a linkage for subsequent functionalization with mesogenic groups. Properties of the NPs were characterized by HRTEM, NMR, DSC, TGA, UV/vis, OPM, and XRD studies. The results confirmed the formation of a stable nematic mesophase above 37.5 °C for NPs in the 6-11 nm size range.

The effect of a computer-based intervention on adult children of alcoholics.

OBJECTIVES: People who grow up with a family member who has a substance use disorder (SUD) are at risk for serious problems, and yet support for family members focuses mainly on the individual with the SUD. Technology may offer a way to make support widely available to family members of those with SUDs. This small randomized trial examined an online system of resources called CHESS (Comprehensive Health Enhancement Support System) for adult children of alcoholics (ACOAs), a population at greater risk for SUDs, depression, and other difficulties than adults whose parents were not alcoholics. METHODS: The study randomized 23 self-identified ACOAs to 3 interventions for 8 weeks. The goal was to increase participants' treatment compliance and psychological health. The interventions were therapy only, CHESS only, and CHESS plus therapy. We used 2 measures: compliance with treatment, gauged by attendance in group therapy for the 2 groups assigned to therapy, and aspects of psychological health or distress, measured by a survey with items from 7 scales. RESULTS: The CHESS-plus-therapy group had an attendance rate in group therapy of 81.5% compared to 42.8% for the therapy-only group. The CHESS-only intervention had the largest effect size on 5 of the 7 measures of psychological health or distress. In 4 of the 5 cases, the effect size was large; in 1 case, it was moderate. CONCLUSIONS: The findings of this pilot study are based on a small sample, but they suggest the need for more research and the potentially important role of technology in behavioral health treatment.

Unexpected phenol production from arylboronic acids under palladium-free conditions; organocatalyzed air oxidation.

An intriguing class of quinones that efficiently catalyze the air oxidation (overall hydroxylation) of arylboronic acids to the corresponding phenol is reported. Autocatalysis in the parent system is particularly efficient and leads to rapid, quantitative synthesis of quinones such as 4 from boronic acid 1 at room temperature using air as stoichiometric oxidant. The efficiency results from a balance between two-stage conjugate addition and migration with each step driven by aromatization of a naphthalene fragment.