The Sialic Acid Binding Protein,Hsa, in Streptococcus gordonii DL1 also Mediates Intergeneric Coaggregation with Veillonella Species

Dental biofilm development involves initial colonization of the tooth’s surface by pioneer colonizers, followed by cell-cell coaggregation between the pioneer and later colonizers. Streptococcus gordonii is one of the pioneer colonizers. In addition to its role in oral biofilm development, S. gordonii also is a pathogen in infective endocarditis in susceptible humans. A surface adhesin, Hsa, has been shown to play a critical role in colonization of S. gordonii on the heart tissue; however, its role in oral biofilm development has not been reported. In this study we demonstrate that Hsa is essential for coaggregation between S. gordonii and Veillonella sp., which are bridging species connecting the pioneer colonizers to the late colonizers. Interestingly, the same domains shown to be required for Hsa binding to sialic acid on the human cell surface are also required for coaggregation with Veillonella sp. However, sialic acid appeared not to be required for this intergeneric coaggregation. This result suggests that although the same domains of Hsa are involved in binding to eukaryotic as well as Veillonella cells, the binding mechanism is different. The gene expression pattern of hsa was also studied and shown not to be induced by coaggregation with Veillonella sp.     

Electroosmotic properties of microfluidic channels composed of poly(dimethylsiloxane)

Microfluidic devices fabricated from polymers exhibit great potential in biological analyses. Poly(dimethylsiloxane) (PDMS) has shown promise as a substrate for rapid prototyping of devices. Despite this, disagreement exists in the literature as to the ability of PDMS to support electroosmotic (EO) flow and the stability of that flow over time. We demonstrate that in low ionic strength solutions near neutral in pH, oxidized PDMS had a four-fold greater EO mobility (μ_eo) compared to native PDMS. The greater μ_eo was maintained irrespective of whether glass or PDMS was used as a support forming one side of the channel. This enhanced μ_eo was preserved as long as the channels were filled with an aqueous solution. Upon exposure of the channels to air, the mobility decreased by a factor of two with a half-life of 9 h. The EO properties of the air-exposed, oxidized PDMS were regenerated by exposure to strong base. High ionic strength, neutral in pH buffers compatible with living eukaryotic cells diminished the EO flow in the oxidized PDMS devices to a much greater extent than in the native PDMS devices. For analyses utilizing intact and living cells, oxidation of PDMS may not be an effective strategy to substantially increase the μ_eo.     

Functional responses between PMP3 small membrane proteins and membrane potential

The Plasma Membrane Proteolipid 3 (PMP3, UPF0057 family in Uniprot) family consists of abundant small hydrophobic polypeptides with two predicted transmembrane helices. Plant homologues were upregulated in response to drought/salt-stresses and yeast deletion mutants exhibited conditional growth defects. We report here abundant expression of Group I PMP3 homologues (PMP3(i)hs) during normal vegetative growth in both prokaryotic and eukaryotic cells, at a level comparable to housekeeping genes, implicating the regular cellular functions. Expression of eukaryotic PMP3(i)hs was dramatically upregulated in response to membrane potential (Vm) variability (Vm_var), whereas PMP3(i)hs deletion-knockdown led to Vm changes with conditional growth defects. Bacterial PMP3(i)h yqaE deletion led to a shift of salt sensitivity; Vm_var alternations with exogenous K⁺ addition downregulated prokaryotic PMP3(i)hs, suggesting [K⁺]-Vm_var axis being a significant feedback element in prokaryotic ionic homeostasis. Remarkably, the eukaryotic homologues functionally suppressed the conditional growth defects in bacterial deletion mutant, demonstrating the conserved cross-kingdom membrane functions by PMP3(i)hs. These data demonstrated a direct reciprocal relationship between PMP3(i)hs expression and Vm differentials in both prokaryotic and eukaryotic cells. Cumulative with PMP3(i)hs ubiquitous abundance, their lipid-binding selectivity and membrane protein colocalization, we propose [PMP3(i)hs]-Vm_var axis as a key element in membrane homeostasis.     

G-quadruplexes in genomes of viruses infecting eukaryotes or prokaryotes are under different selection pressures from hosts

G-quadruplexes in viral genomes can be applied as the targets of antiviral therapies, which has attracted wide interest. However, it is still not clear whether the pervasive number of such elements in the viral world is the result of natural selection for functionality. In this study, we identified putative quadruplex-forming sequences (PQSs) across the known viral genomes and analyzed the abundance, structural stability, and conservation of viral PQSs. A Viral Putative G-quadruplex Database (http://jsjds.hzau.edu.cn/MBPC/ViPGD/index.php/home/index) was constructed to collect the details of each viral PQS, which provides guidance for selecting the desirable PQS. The PQS with two putative G-tetrads (G₂-PQS) was significantly enriched in both eukaryotic viruses and prokaryotic viruses, whereas the PQSs with three putative G-tetrads (G₃-PQS) were only enriched in eukaryotic viruses and depleted in prokaryotic viruses. The structural stability of PQSs in prokaryotic viruses was significantly lower than that in eukaryotic viruses. Conservation analysis showed that the G₂-PQS, instead of G₃-PQS, was highly conserved within the genus. This suggested that the G₂-quadruplex might play an important role in viral biology, and the difference in the occurrence of G-quadruplex between eukaryotic viruses and prokaryotic viruses may result from the different selection pressures from hosts.     

Rotation,Structure, and Classification of Prokaryotic V-ATPase

The prokaryotic V-type ATPase/synthases (prokaryotic V-ATPases) have simpler subunit compositions than eukaryotic V-ATPases, and thus are useful subjects for studying chemical, physical and structural properties of V-ATPase. In this review, we focus on the results of recent studies on the structure/function relationships in the V-ATPase from the eubacterium Thermus thermophilus. First, we describe single-molecule analyses of T. thermophilus V-ATPase. Using the single-molecule technique, it was established that the V-ATPase is a rotary motor. Second, we discuss arrangement of subunits in V-ATPase. Third, the crystal structure of the C-subunit (homolog of eukaryotic d-subunit) is described. This funnel-shape subunit appears to cap the proteolipid ring in the V₀ domain in order to accommodate the V₁ central stalk. This structure seems essential for the regulatory reversible association/dissociation of the V₁ and the V₀ domains. Last, we discuss classification of the V-ATPase family. We propose that the term prokaryotic V-ATPases should be used rather than the term archaeal-type ATPase (A-ATPase).     

Calcite and dolomite dissolution rates in the context of microbe–mineral surface interactions

Although microbes have been shown to alter the dissolution rate of carbonate minerals, a mechanistic understanding of the consequences of microbial surface colonization on carbonate dissolution has yet to be achieved. Here we report the use of vertical scanning interferometry (VSI) to study the effect of Shewanella oneidensis MR‐1 surface colonization on the dissolution rates of calcite (CaCO₃) and dolomite (CaMg(CO₃)₂) through qualitative analysis of etch pit development and quantitative measurements of surface‐normal dissolution rates. By quantifying and comparing the significant processes occurring at the microbe–mineral interface, the dominant mechanism of mineral dissolution during surface colonization was determined. MR‐1 attachment under aerobic conditions was found to influence carbonate dissolution through two distinct mechanistic pathways: (1) inhibition of carbonate dissolution through interference with etch pit development and (2) excavation of carbonate material at the cell–mineral interface during irreversible attachment to the mineral surface. The relative importance of these two competing effects was found to vary with the solubility of the carbonate mineral studied. For the faster‐dissolving calcite substrates, inhibition of dissolution by attachment and subsequent extracellular polysaccharide (EPS) production was the dominant effect associated with MR‐1 surface colonization. This interference with etch pit development resulted in a 40–70% decrease in the surface normal dissolution rate relative to cell‐free controls, depending primarily on the concentration of cells in solution. However, in the case of the slower‐dissolving dolomite substrates, carbonate material displaced during the entrenchment of cells on the surface far outweighed the abiotic dissolution rate. Therefore, during the initial stages of surface colonization, dolomite dissolution rates were actually enhanced by MR‐1 attachment. This study demonstrates the dynamic and competitive relationship between microbial surface colonization and mineral dissolution that may be expected to occur in natural environments.     

The amino acid sequences of the cytochromes c553 from Porphyridium cruentum and Aphanizomenon flos-aquae

The amino acid sequences of cytochrome c₅₅₃ from the eukaryotic red alga Porphyridium cruentum and from the prokaryotic cyanobacterium Aphanizomenon flos-aquae have been determined from the tryptic and cyanogen bromide peptides. The results indicate that a charged region of these proteins has evolved with special rapidity to accomodate a rapid evolution of a binding site in the P₇₀₀ electron acceptor complex.     

Functional characterization of a Na+-coupled dicarboxylate transporter from Bacillus licheniformis

The Na⁺-coupled dicarboxylate transporter, SdcL, from Bacillus licheniformis is a member of the divalent anion/Na⁺ symporter (DASS) family that includes the bacterial Na⁺/dicarboxylate cotransporter SdcS (from Staphyloccocus aureus) and the mammalian Na⁺/dicarboxylate cotransporters, NaDC1 and NaDC3. The transport properties of SdcL produced in Escherichia coli are similar to those of its prokaryotic and eukaryotic counterparts, involving the Na⁺-dependent transport of dicarboxylates such as succinate or malate across the cytoplasmic membrane with a K_m of ～ 6 μM. SdcL may also transport aspartate, α-ketoglutarate and oxaloacetate with low affinity. The cotransport of Na⁺ and dicarboxylate by SdcL has an apparent stoichiometry of 2:1, and a K_0.5 for Na⁺ of 0.9 mM. Our findings represent the characterization of another prokaryotic protein of the DASS family with transport properties similar to its eukaryotic counterparts, but with a broader substrate specificity than other prokaryotic DASS family members. The broader range of substrates carried by SdcL may provide insight into domains of the protein that allow a more flexible or larger substrate binding pocket.     

Reduction of the Inflammatory Responses against Alginate-Poly-L-Lysine Microcapsules by Anti-Biofouling Surfaces of PEG-b-PLL Diblock Copolymers

Large-scale application of alginate-poly-L-lysine (alginate-PLL) capsules used for microencapsulation of living cells is hampered by varying degrees of success, caused by tissue responses against the capsules in the host. A major cause is proinflammatory PLL which is applied at the surface to provide semipermeable properties and immunoprotection. In this study, we investigated whether application of poly(ethylene glycol)-block-poly(L-lysine hydrochloride) diblock copolymers (PEG-b-PLL) can reduce the responses against PLL on alginate-matrices. The application of PEG-b-PLL was studied in two manners: (i) as a substitute for PLL or (ii) as an anti-biofouling layer on top of a proinflammatory, but immunoprotective, semipermeable alginate-PLL₁₀₀ membrane. Transmission FTIR was applied to monitor the binding of PEG-b-PLL. When applied as a substitute for PLL, strong host responses in mice were observed. These responses were caused by insufficient binding of the PLL block of the diblock copolymers confirmed by FTIR. When PEG-b-PLL was applied as an anti-biofouling layer on top of PLL₁₀₀ the responses in mice were severely reduced. Building an effective anti-biofouling layer required 50 hours as confirmed by FTIR, immunocytochemistry and XPS. Our study provides new insight in the binding requirements of polyamino acids necessary to provide an immunoprotective membrane. Furthermore, we present a relatively simple method to mask proinflammatory components on the surface of microcapsules to reduce host responses. Finally, but most importantly, our study illustrates the importance of combining physicochemical and biological methods to understand the complex interactions at the capsules'' surface that determine the success or failure of microcapsules applicable for cell-encapsulation.     

Diversity and stability of coral endolithic microbial communities at a naturally high pCO2 reef

The health and functioning of reef‐building corals is dependent on a balanced association with prokaryotic and eukaryotic microbes. The coral skeleton harbours numerous endolithic microbes, but their diversity, ecological roles and responses to environmental stress, including ocean acidification (OA), are not well characterized. This study tests whether pH affects the diversity and structure of prokaryotic and eukaryotic algal communities associated with skeletons of Porites spp. using targeted amplicon (16S rRNA gene, UPA and tufA) sequencing. We found that the composition of endolithic communities in the massive coral Porites spp. inhabiting a naturally high pCO₂ reef (avg. pCO₂ 811 μatm) is not significantly different from corals inhabiting reference sites (avg. pCO₂ 357 μatm), suggesting that these microbiomes are less disturbed by OA than previously thought. Possible explanations may be that the endolithic microhabitat is highly homeostatic or that the endolithic micro‐organisms are well adapted to a wide pH range. Some of the microbial taxa identified include nitrogen‐fixing bacteria (Rhizobiales and cyanobacteria), algicidal bacteria in the phylum Bacteroidetes, symbiotic bacteria in the family Endozoicomoniaceae, and endolithic green algae, considered the major microbial agent of reef bioerosion. Additionally, we test whether host species has an effect on the endolithic community structure. We show that the endolithic community of massive Porites spp. is substantially different and more diverse than that found in skeletons of the branching species Seriatopora hystrix and Pocillopora damicornis. This study reveals highly diverse and structured microbial communities in Porites spp. skeletons that are possibly resilient to OA.     

Glycerolipid synthesis in Chlorella kessleri 11h: I. Existence of a eukaryotic pathway

The fatty acid distributions at the sn-1 and sn-2 positions in major chloroplast lipids of Chlorella kessleri 11h, monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG), were determined to show the coexistence of both C₁₆ and C₁₈ acids at the sn-2 position, i.e. of prokaryotic and eukaryotic types in these galactolipids. For investigation of the biosynthetic pathway for glycerolipids in C. kessleri 11h, cells were fed with [¹⁴C]acetate for 30 min, and then the distribution of the radioactivity among glycerolipids and their constituent fatty acids during the subsequent chase period was determined. MGDG and DGDG were labeled predominantly as the sn-1-C₁₈-sn-2-C₁₆ (C₁₈/C₁₆) species as early as by the start of the chase, which suggested the synthesis of these lipids within chloroplasts via a prokaryotic pathway. On the other hand, the sn-1-C₁₈-sn-2-C₁₈ (C₁₈/C₁₈) species of these galactolipids gradually gained radioactivity at later times, concomitant with a decrease in the radioactivity of the C₁₈/C₁₈ species of phosphatidylcholine (PC). The change at later times can be explained by the conversion of the C₁₈/C₁₈ species of PC into galactolipids through a eukaryotic pathway. The results showed that C. kessleri 11h, distinct from most of other green algal species that were postulated mainly to use a prokaryotic pathway for the synthesis of chloroplast lipids, is similar to a group of higher plants designated as 16:3 plants in terms of the cooperation of prokaryotic and eukaryotic pathways to synthesize chloroplast lipids. We propose that the physiological function of the eukaryotic pathway in C. kessleri 11h is to supply chloroplast membranes with 18:3/18:3-MGDG for their functioning, and that the acquisition of a eukaryotic pathway by green algae was favorable for evolution into land plants.     

Immobilization of silver in polypropylene membrane for anti-biofouling performance

In this study, a method was developed to immobilize silver onto polypropylene (PP) membrane surfaces for improved anti-biofouling performance. A commercial PP membrane was first grafted with the thiol functional groups, and then silver ions were immobilized onto the PP membrane surface through coordinating with the thiol groups. The immobilized silver was found to be very stable, with only ～1.1% of the immobilized silver being leached out during a leaching test. The surface of the modified membrane (PPS-Ag) was examined with ATR-FTIR and XPS analysis, which verified the successful grafting of the thiol groups and the coordination of silver ions on the membrane surface. The surface properties of the membrane were also characterized by SEM, AFM and water contact angle measurements. The PPS-Ag membrane was found to have a smoother and more hydrophilic surface than the PP membrane. Both Gram-negative bacteria, Escherichia coli, and Gram-positive bacteria, Staphylococcus aureus, were used to evaluate the antibacterial and anti-biofouling performance of the PPS-Ag membrane. From disk diffusion experiments, the PPS-Ag membrane exhibited the capability of inhibiting the growth of both the Gram-negative and Gram-positive bacteria tested. The anti-biofouling performance of the membrane was assessed by immersion in a mixed suspension of E. coli and S. aureus and filtration tests. The PPS-Ag membrane showed a stable and significantly enhanced anti-biofouling performance as compared with the PP membrane. The results in this study demonstrate that biofouling of a PP membrane can be sufficiently overcome through immobilizing silver onto the membrane surface.     

Microbial landscapes on the outer tissue surfaces of the reef-building coral <Emphasis Type="Italic">Porites compressa</Emphasis>

Microbial-coral interactions are increasingly recognized as important for coral health and disease. Visualizing these interactions is important for understanding where, when, and how the coral animal and microbes interact. Porites compressa, preserved using Parducz fixative and examined by scanning electron microscopy, revealed a changing microbial landscape. The external cell layers of this coral were invariably clean of directly adhering microbes, unlike coral-associated mucus. In colonies with expanded polyps, secreted mucus rapidly dissipated, although blobs of new mucus were common; the coral epidermal cells expressed cilia, which are presumably used to clean the surface, and coral-associated microbes were present as flocs, possibly enmeshed in mucus. In colonies with permanently contracted polyps, the coral epidermis had lost cilia and a stable, multi-lamellar mucous sheet covered the surface of the animal. This sheet became heavily colonized by both prokaryotic and eukaryotic microbes, however these microbes did not penetrate the mucous sheet and the animal’s epidermal cell surfaces remained sterile. These observations show that relationships between this coral animal and associated microbes are highly dynamic.     

Improved antifouling properties of PVDF membranes modified with oppositely charged copolymer

Biofouling resulting from the attachment of microorganisms communities to the membrane surface is the major obstacle for the widespread application of membrane technology. This work develops a feasible approach to prepare an anti-biofouling poly(vinylidene fluoride) (PVDF) membrane. A copolymer that possessed oppositely charged groups was first synthesized via radical copolymerization with methyl methacrylate, 2-methacryloxy ethyltrimethyl ammonium chloride and 2-acrylamide-2-methyl propane sulphonic acid as monomers. The copolymer was blended with the PVDF powder to prepare the antifouling membrane via the immersed phase inversion method. The antifouling properties of the modified PVDF membrane were studied by X-ray photoelectron spectroscopy, field emission scanning electron microscopy, water contact angle measurement, zeta-potential measurement, protein adsorption, microbial adhesion and filtration experiments. The modified PVDF membrane showed limited adsorption and adhesion of protein bovine serum albumin and microbes (Escherichia coli and Saccharomyces cerevisiae) with increasing copolymer concentration in the casting solution. The modified PVDF membrane exhibited excellent antibiofouling properties.     

Spectroscopic and thermodynamic evidence for antimicrobial peptide membrane selectivity

In our laboratory we developed a series of antimicrobial peptides that exhibit selectivity and potency for prokaryotic over eukaryotic cells (Hicks et al., 2007). Circular dichroism (CD), isothermal calorimetry (ITC) and calcein leakage assays were conducted to determine the mechanism of lipid binding of a representative peptide 1 (Ac-GF-Tic-Oic-GK-Tic-Oic-GF-Tic-Oic-GK-Tic-KKKK-CONH₂) to model membranes. POPC liposomes were used as a simple model for eukaryotic membranes and 4:1 POPC:POPG liposomes were used as a simple model for prokaryotic membranes. CD, ITC and calcein leakage data clearly indicate that compound 1 interacts via very different mechanisms with the two different liposome membranes. Compound 1 exhibits weaker binding and induces less calcein leakage in POPC liposomes than POPC:POPG (4:1 mole ratio) liposomes. The predominant binding mechanism to POPC appears to be limited to surface interactions while the mechanism of binding to 4:1 POPC:POPG most likely involves some type of pore formation.     

Fecal microbiota transplantation prevents Candida albicans from colonizing the gastrointestinal tract

Gut microbes symbiotically colonize the gastrointestinal (GI) tract, interacting with each other and their host to maintain GI tract homeostasis. Recent reports have shown that gut microbes help protect the gut from colonization by pathogenic microbes. Here, we report that commensal microbes prevent colonization of the GI tract by the pathogenic fungus, Candida albicans. Wild‐type specific pathogen‐free (SPF) mice are resistant to C. albicans colonization of the GI tract. However, administering certain antibiotics to SPF mice enables C. albicans colonization. Quantitative kinetics of commensal bacteria are inversely correlated with the number of C. albicans in the gut. Here, we provide further evidence that transplantation of fecal microbiota is effective in preventing Candida colonization of the GI tract. These data demonstrate the importance of commensal bacteria as a barrier for the GI tract surface and highlight the potential clinical applications of commensal bacteria in preventing pathogenic fungal infections.     

Symbiotic lactobacilli stimulate gut epithelial proliferation via Nox‐mediated generation of reactive oxygen species

The resident prokaryotic microbiota of the metazoan gut elicits profound effects on the growth and development of the intestine. However, the molecular mechanisms of symbiotic prokaryotic–eukaryotic cross‐talk in the gut are largely unknown. It is increasingly recognized that physiologically generated reactive oxygen species (ROS) function as signalling secondary messengers that influence cellular proliferation and differentiation in a variety of biological systems. Here, we report that commensal bacteria, particularly members of the genus Lactobacillus, can stimulate NADPH oxidase 1 (Nox1)‐dependent ROS generation and consequent cellular proliferation in intestinal stem cells upon initial ingestion into the murine or Drosophila intestine. Our data identify and highlight a highly conserved mechanism that symbiotic microorganisms utilize in eukaryotic growth and development. Additionally, the work suggests that specific redox‐mediated functions may be assigned to specific bacterial taxa and may contribute to the identification of microbes with probiotic potential.     

Pivotal and distinct role for Plasmodium actin capping protein alpha during blood infection of the malaria parasite

Accurate regulation of microfilament dynamics is central to cell growth, motility and response to environmental stimuli. Stabilizing and depolymerizing proteins control the steady‐state levels of filamentous (F‐) actin. Capping protein (CP) binds to free barbed ends, thereby arresting microfilament growth and restraining elongation to remaining free barbed ends. In all CPs characterized to date, alpha and beta subunits form the active heterodimer. Here, we show in a eukaryotic parasitic cell that the two CP subunits can be functionally separated. Unlike the beta subunit, the CP alpha subunit of the apicomplexan parasite Plasmodium is refractory to targeted gene deletion during blood infection in the mammalian host. Combinatorial complementation of Plasmodium berghei CP genes with the orthologs from Plasmodium falciparum verified distinct activities of CP alpha and CP alpha/beta during parasite life cycle progression. Recombinant Plasmodium CP alpha could be produced in Escherichia coli in the absence of the beta subunit and the protein displayed F‐actin capping activity. Thus, the functional separation of two CP subunits in a parasitic eukaryotic cell and the F‐actin capping activity of CP alpha expand the repertoire of microfilament regulatory mechanisms assigned to CPs.     

Soil prokaryotic community shows no response to 2 years of simulated nitrogen deposition in an arid ecosystem in northwestern China

An arid ecosystem might be sensitive to nitrogen (N) deposition, but the associated ecosystem-specific response of soil microbes is not well studied. To assess the N enrichment effects on plant and prokaryotic community diversity, we performed a two-year NH₄NO₃ treatment in a desert steppe in northwestern China. Results showed that N addition increased plant aboveground biomass and decreased plant Shannon diversity. A C₄ herb (Salsola collina) became dominant, and loss of legume species was observed. The concentrations of soil NH₄⁺-N, NO₃⁻-N, microbial biomass N, and the plant aboveground biomass N pool increased in contrast to total N, suggesting that the N input into the arid ecosystem might mainly be assimilated by plants and exit the ecosystem. Remarkably, the α-diversity and structure of the soil prokaryotic community did not vary even at the highest N addition rate. Structural equation modelling further found that the plant aboveground N pool counteracted the acidification effect of N deposition and maintained soil pH thus partially stabilizing the composition of prokaryotic communities in a desert steppe. These findings suggested that the plants and N loss might contribute to the lack of responsiveness of soil prokaryotic community to N deposition in a desert steppe.     

Polydimethylsiloxane Films Modified with Chitosan/Pectin Multilayers as Scaffolds for Mesenchymal Stem Cells

Solid (smooth) and porous films of polydimethylsiloxane (PDMS) have been obtained; the effect of their structure on the adhesion of mesenchymal stem cells (MSCs) on their surface was found. It is shown that modification of these films with a (chitosan/pectin)₄ multilayer increased the efficiency of viable cell adhesion. A (3-aminopropyl)triethoxysilane–glutaraldehyde system was used to enhance the binding of the polysaccharide layer to the hydrophobic surface of PDMS. It was found that MSCs formed a monolayer culture of the fibroblast-like cells with high viability on porous PDMS modified with (chitosan/pectin)₄.