Migration In Electrophoresis Research Articles

Molecular characterization of histone gene in golden pompano (Trachinotus ovatus) and antimicrobial activity of its derived peptides

In addition to controlling gene expression, mediating DNA folding into chromatin, and responding to immunological stimuli, histones are also thought to have antimicrobial effects. This study identified the molecular characteristics of core Histone MacroH2A2 (TOMacroH2A2) and Histone H2B 1/2 (TOH2B) from Trachinotus ovatus, and the antimicrobial potential of their derived peptides (To.mh2a and To. h2b). The open reading frames (ORFs) of TOMacroH2A2 and TOH2B from T. ovatus were 1010 bp and 375 bp, encoding polypeptides of 369 and 124 amino acids, respectively. The TOMacroH2A2 included an H2A domain and an A1pp domain, while TOH2B included an H2B domain. The amino acid sequences of TOMacroH2A2 and TOH2B demonstrated high homology with other teleost's sequences of histone macroh2a2 and histone h2b, with homologies exceeding 90 %. Expression analysis showed high expression of TOMacroH2A2 in brain, stomach, heart, and skin tissues and TOH2B in gill, brain, and skin tissues. In addition, the histone-derived peptides To. mh2a and To. h2b, synthesized based on two histone sequences from T. ovatus, exhibited typical physical characteristics of antimicrobial peptides, including positive charges, amphipathicity, hydrophobicity, and rich α-helix structure. Crucially, the vitro antibacterial results demonstrated that To. mh2a and To. h2b can inhibit the growth of various aquatic pathogens including Streptococcus agalactiae, Staphylococcus aureus, Bacillus subtilis, Acinetobacter baumannii, Aeromonas hydrophila, and Escherichia coli to varying degrees. Specifically, To. mh2a and To. h2b were capable of disrupting the cell surface structures of S. aureus and penetrating the cell membrane, leading to the leakage of cellular contents, thereby exerting their antibacterial effects. Furthermore, gel electrophoresis migration assays showed that To. mh2a and To. h2b participated in antimicrobial activity by binding to bacterial genomic DNA and reducing the migration rate of gDNA in a dose-dependent manner. The minimum effective concentration for binding to DNA was approximately 50 μM. In conclusion, our study suggested that To. mh2a and To. h2b can act as antimicrobial peptides, providing a potential strategy for controlling bacterial diseases in T. ovatus.

Electroneutral Layer Dynamics Drive Ion Migration in Low Frequency AC Electrophoresis Below the Water Electrolysis Threshold.

Low-frequency AC electrophoresis lies in a regime between DC microchannel electrophoresis and dielectrophoresis, which typically utilizes frequencies above 1000 Hz. Although few electrophoretic methods have been reported in this ≤100 Hz range, traveling wave electrophoresis (TWE) and transverse AC electrophoresis (TrACE) operate in this frequency range, and use low voltages to avoid bubble formation from water electrolysis. TWE provides molecular separations with enhanced control and TrACE provides multiplexed, multiparameter particle characterization. However, two related fundamental questions remain about the mechanisms of electrophoretic migration in these systems. First, particle electrophoresis in TrACE is largely captured by a simple model that combines the alternating electric field with DC electrokinetics, but a deviation from the model is observed for applied square electric field waves that increases with decreasing frequency. Second, although electrode charging is believed to drive ion migration in TWE, the estimated electrode charging time is about 2-3 orders of magnitude faster than the wave period. In this study, a 1D finite numerical model that excludes Faradaic reactions simulates ion and particle migration across the microchannel width in TrACE. The 1D model results show good agreement with both particle and ion migration in TrACE systems. Furthermore, although ion migration between the pair of electrodes slows during each excursion of a 1 Hz square wave, there is substantial ion migration throughout the 0.5 s half-period. This modeling result agrees with experimental observations in TWE. Therefore, the clarification of the mechanisms of ion migration in these low-frequency and low-voltage AC electrophoresis is expected to expand their applications.

Enzyme variants in biosynthesis and biological assessment of different molecular weight hyaluronan

In the present study, low- and high-molecular-weight hyaluronic acids (LMW-HA and HMW-HA) were synthesized in vitro by truncated Streptococcus equisimilis hyaluronan synthases (SeHAS). The enzyme kinetic parameters were determined for each enzyme variant. The MW, structure, dispersity, and biological activity of polymers were determined by electrophoresis, FTIR spectroscopy, carbazole, cell proliferation, and cell migration assay, respectively. The specific activities were calculated as 7.5, 6.8, 4.9, and 2.8 µgHA µgenzyme−1 min−1 for SeHAS, HAS123, HAS23, and HASIntra, respectively. The results revealed SeHAS produced a polydisperse HMW-HA (268 kDa), while HAS123 and HAS23 produced a polydisperse LMW-HA (< 30 kDa). Interestingly, HASIntra produced a low-disperse LMW-HA. Kinetics studies revealed the truncated variants displayed increased Km values for two substrates when compared to the wild-type enzyme. Biological assessments indicated all LMW-HAs showed a dose-dependent proliferation activity on endothelial cells (ECs), whereas HMW-HAs exhibited an inhibitory effect. Also, LMW-HAs had the highest cell migration effect at 10 µg/mL, while at 200 µg/mL, both LMW- and HMW-HAs postponed the healing recovery rate. The study elucidated that the transmembrane domains (TMDs) of SeHAS affect the enzyme kinetics, HA-titer, HA-size, and HA-dispersity. These findings open new insight into the rational engineering of SeHAS to produce size-defined HA.Graphical abstract

STM1863, a Member of the DUFs Protein Family, Is Involved in Environmental Adaptation, Biofilm Formation, and Virulence in Salmonella Typhimurium.

Salmonella Typhimurium (STM) is an important zoonotic Gram-negative pathogen that can cause infection in a variety of livestock and poultry. Meanwhile, as an important foodborne pathogen, the bacterium can survive in various stressful environments and transmits through the fecal-oral route, posing a serious threat to global food safety. To investigate the roles of STM1863, a member of the DUFs protein family, involved in STM environmental adaptation, biofilm formation, and virulence. We analyzed the molecular characteristics of the protein encoded by STM1863 gene and examined intra- and extracellular expression levels of STM1863 gene in mouse macrophages. Furthermore, we constructed STM1863 gene deletion and complementation strains and determined its environmental adaptation under stressful conditions such as acid, alkali, high salt, bile salt, and oxidation. And the capacity of biofilm formation and pathogenicity of those strains were analyzed and compared. In addition, the interaction between the promoter of STM1863 gene and RcsB protein was analyzed using DNA gel electrophoresis migration assay (electrophoretic mobility shift assay [EMSA]). The experiments revealed that acid adaptability and biofilm formation ability of STM1863 gene deletion strain were significantly weakened compared with the parental and complementary strains. Moreover, the adhesion and invasion ability of STM1863 deletion strain to mouse macrophages was significantly decreased, while the median lethal dose (LD50) increased by 2.148-fold compared with the parental strain. In addition, EMSA confirmed that RcsB protein could bind to the promoter sequence of STM1863 gene, suggesting that the expression of STM1863 gene might be modulated by RcsB. The present study demonstrated for the first time that STM1863, a member of the DUFs protein family, is involved in the modulation of environmental adaptation, biofilm formation, and virulence.

Antioxidant and antiproliferative effect of a glycosaminoglycan extract from Rapana venosa marine snail.

Marine glycosaminoglycans (GAG) isolated from different invertebrates, such as molluscs, starfish or jellyfish, have been described as unique molecules with important pharmacological applications. Scarce information is available on GAG extract from Rapana venosa marine snail. The aim of this study was to isolate a GAG extract from R. venosa marine snail and to investigate its physicochemical, antioxidant and antiproliferative properties for further biomedical use. The morphology, chemical and elemental composition of the extract were established as well as the sulfate content and N- to O-sulfation ratio. Fourier transform infrared (FTIR) spectra indicated that GAG extract presented similar structural characteristics to bovine heparan sulfate and chondroitin sulfate. The pattern of extract migration in agarose gel electrophoresis and specific digestion with chondroitinase ABC and heparinase III indicated the presence of a mixture of chondroitin sulfate-type GAG, as main component, and heparan sulfate-type GAG. Free radical scavenging and ferric ion reducing assays showed that GAG extract had high antioxidant activity, which slightly decreased after enzymatic treatment. In vitro MTT and Live/Dead assays showed that GAG extract had the ability to inhibit cell proliferation in human Hep-2 cell cultures, at cytocompatible concentrations in normal NCTC clone L929 fibroblasts. This capacity decreased after enzymatic digestion, in accordance to the antioxidant activity of the products. Tumoral cell migration was also inhibited by GAG extract and its digestion products. Overall, GAG extract from R. venosa marine snail exhibited antioxidant and antiproliferative activities, suggesting its potential use as novel bioactive compound for biomedical applications.

Effect of cell shape on nonlinear electrophoresis migration

This study contributes to the renewed interest in the study of nonlinear electrophoresis of colloidal particles. In this work the influence of cell shape on electrophoretic migration under the nonlinear regimes of moderate and strong field regimes was assessed. Four types of bacterial and yeast cells (one spherical, three non-spherical) were studied and their electrophoretic mobilities for the moderate and strong electric field magnitude regimes were estimated experimentally. The parameter of sphericity was employed to assess the effect cell shape on the nonlinear electrophoresis migration velocity and corresponding mobility under the two electric field magnitude regimes studied. As particle migration under nonlinear electrophoresis depends on particle size and shape, the results in terms of mobilities of nonlinear electrophoresis were presented as function of cell hydrodynamic diameter and sphericity. The results indicated that the magnitude of the mobilities of nonlinear electrophoresis for cells increase with increasing cell size and increase with increasing deviations from spherical shape, which is indicated by lower sphericity values. The results presented here are the very first assessment of the two types of mobilities of nonlinear electrophoresis of cells as a function of size and shape.

Particle mass transport in impact electrochemistry

Impact electrochemistry is a set of methods in which individual micro- or nanoscale particles are detected and analyzed at a miniaturized electrode. Quantitative interpretation of the results, in particular the determination of ultralow concentrations, relies heavily on modeling the mass transport of the particles being analyzed. This is particularly subtle since, due to favorable scaling with increasing particle size, migration and convection play a disproportionate role in the transport of such particles compared to that of small molecules. Here we summarize the main governing principles in electrochemically-driven particle transport. We particularly emphasize the difference between particle electrophoresis and small-ion migration, which has led to inaccuracies in the recent literature.

Circular RNA migration in agarose gel electrophoresis

Circular RNAs are garnering increasing interest as potential regulatory RNAs and a format for gene expression. The characterization of circular RNA using analytical techniques commonly employed in the literature, such as gel electrophoresis, can, under differing conditions, yield different results when attempting to distinguish circular RNA from linear RNA of similar molecular weights. Here, we describe circular RNA migration in different conditions, analyzed by gel electrophoresis and high-performance liquid chromatography (HPLC). We characterize key parameters that affect the migration pattern of circular RNA in gel electrophoresis systems, which include gel type, electrophoresis time, sample buffer composition, and voltage. Finally, we demonstrate the utility of orthogonal analytical tests for circular RNA that take advantage of its covalently closed structure to further distinguish circular RNA from linear RNA following invitro synthesis.

Characterization of a 36 kDa antigenic protein of fish-specific monoclonal-antibody 8F5

The 36 kDa antigenic protein of a fish-specific monoclonal antibody (mAb), 8F5, previously developed to detect fish in foodstuffs to protect fish allergic individuals, was characterized to establish its identity and to identify the fish-specific epitope. We hypothesized that this antigenic protein is tropomyosin based on its thermal stability and molecular weight. Western blot showed that both the 36 kDa protein and fish tropomyosin were recognized by mAb 8F5, and their molecular weight migration in urea gel electrophoresis was identical. In addition to matching the amino acid composition profile, this 36 kDa protein’s sequences precisely correspond to those in fish tropomyosin fragments. Further analysis revealed the sequence of the fish-specific epitope bound by mAb 8F5 to be EDDLVALQKK. These results confirm that the 36 kDa protein is indeed tropomyosin and will be a suitable biomarker for the immunodetection of fish in cooked food.

Development of a rapid electrophoretic assay for genomic DNA damage quantification

Accuracy, sensitivity, simplicity, reproducibility, and low-cost are desirable requirements for genotoxicity assessment techniques. Here we describe a simple electrophoretic assay for genomic DNA lesions quantification (EAsy-GeL) based on subjecting DNA samples to rapid unwinding/renaturation treatments and neutral agarose gel electrophoresis. The experiments performed in this work involved different biological samples exposed to increasing environmental-simulated doses of ultraviolet-B (UVB) radiation, such as Escherichia coli, human leukocytes, and isolated human genomic DNA. DNA extraction was carried out using a universal and low-cost protocol, which takes about 4 h. Before electrophoresis migration, DNA samples were kept into a neutral buffer to detect double-strand breaks (DSBs) or subjected to a 5-min step of alkaline unwinding and neutral renaturation to detect single-strand breaks (SSBs) or incubated with the DNA repair enzyme T4-endonuclease V for the detection of cyclobutane pyrimidine dimers (CPDs) before the 5-min step of DNA unwinding/renaturation. Then, all DNA samples were separated by neutral agarose gel electrophoresis, the DNA average length of each lane was calculated through the use of free software, and the frequency of DNA breaks per kbp was determined by a simple rule of three. Dose-response experiments allowed the quantification of different levels of DNA damage per electrophoretic run, varying from a constant and low amount of DSBs/SSBs to high and dose-dependent levels of CPDs. Compared with other assays based on alkaline unwinding and gel electrophoresis, EAsy-GeL has important advantages for both environmental monitoring and laboratory testing purposes. The simplicity and applicability of this protocol to other types of DNA lesions, biological models, and agents make it ideal for genotoxicity, DNA repair studies, as well as for assessing exposure risks to ecosystems and human health.

Dumbbell kinetic theory for polymers in a combination of flow and external electric field.

Combining Poiseuille flow with an external electric field is a demonstrated method to drive transverse migration in capillary electrophoresis. Despite both computational and experimental studies, a number of questions about how to best model polymers under these conditions remains. Attempts have been made to develop a kinetic theory for a bead-spring dumbbell model, but these have only been accurate at low electric field strength and have not captured the nonmonotonic relationship between migration and electric field strength. In this paper, we revisit the development of a kinetic theory for a bead-spring dumbbell in a combination of parabolic flow and an external electric field. The resultant theory yields a compact formula that predicts polymer concentration profiles that agree excellently with our Brownian dynamics simulations including the aforementioned nonmonotonic relationship. Furthermore, we compare our theoretical results to experimental data and find that our model nearly quantitatively predicts the position of the maximum in migration.

Coupling of capillary electrophoresis with electrospray ionization multiplexing ion mobility spectrometry.

In this work, ion mobility spectrometry (IMS) function as a detector and another dimension of separation was coupled with CE to achieve two-dimensional separation. To improve the performance of hyphenated CE-IMS instrument, electrospray ionization correlation ion mobility spectrometry is evaluated and compared with traditional signal averaging data acquisition method using tetraalkylammonium bromide compounds. The effect of various parameters on the separation including sample introduction, sheath fluid of CE and drift gas, data acquisition method of IMS were investigated. The experimental result shows that the optimal conditions are as follows: hydrodynamic sample injection method, the electrophoresis voltage is 10 kilo volts, 5 mmol/L ammonium acetate buffer solution containing 80% acetonitrile as both the background electrolyte and the electrospray ionization sheath fluid, the ESI liquid flow rate is 4.5 μL/min, the drift voltage is 10.5 kilo volts, the drift gas temperature is 383 K and the drift gas flow rate is 300 mL/min. Under the above conditions, the mixture standards of seven tetraalkylammoniums can be completely separated within 10 min both by CE and IMS. The linear range was 5-250 μg/mL, with LOD of 0.152, 0.204, 0.277, 0.382, 0.466, 0.623 and 0.892 μg/mL, respectively. Compared with traditional capillary electrophoresis detection methods, the developed CE-ESI-IMS method not only provide two sets of qualitative parameters including electrophoresis migration time and ion drift time, ion mobility spectrometer can also provide an additional dimension of separation and could apply to the detection ultra-violet transparent compounds or none fluorescent compounds.

Multiplex PCR to simultaneous identification of five traditional Chinese medicinal seahorses

Seahorse is one the most commonly used medicinal animal in China. Five species of Hippocampus are recorded as seahorse in the Chinese Pharmacopoeia. Because of the rapid decrease, several other Hippocampus species are often adulterants as medicinal seahorse in the herbal market, which compromise clinical efficacy and pose threat to endangered seahorse species conversation. Herein, a multiplex polymerase chain reaction (mPCR) method was developed to identify the biological sources of medicinal seahorses.Based on the sequences of mitochondrial DNA, five specific primers for Hippocampus trimaculatus, H. kelloggi, H. kuda, H. histrix and H. mohnikei (H. japonicus)were designed, respectively. Multiplex PCR yields the products of 155, 222, 292, 352, 458 bp amplicons in the present of DNA templates of H. kuda, H. mohnikei, H. kelloggi, H. histrix and H. trimaculatus, respectively. This multiplex PCR method which electrophoresis migration of different lengths of DNA bands allowed simultaneous identification of all the five medicinal seahorses in a single assay. It showed that this multiplex PCR assay is useful for the simultaneous identification the biological sources of complex multi-source samples, which could provide a useful tool for the quality control of seahorses.

Revisit of wall-induced lateral migration in particle electrophoresis through a straight rectangular microchannel: Effects of particle zeta potential.

Previous studies have reported a lateral migration in particle electrophoresis through a straight rectangular microchannel. This phenomenon arises from the inherent wall-induced electrical lift that can be exploited to focus and separate particles for microfluidic applications. Such a dielectrophoretic-like force has been recently found to vary with the buffer concentration. We demonstrate in this work that the particle zeta potential also has a significant effect on the wall-induced electrical lift. We perform an experimental study of the lateral migration of equal-sized polystyrene particles with varying surface charges under identical electrokinetic flow conditions. Surprisingly, an enhanced focusing is observed for particles with a faster electrokinetic motion, which indicates a substantially larger electrical lift for particles with a smaller zeta potential. We speculate this phenomenon may be correlated with the particle surface conduction that is a strong function of particle and fluid properties.

Precision of CAPILLARYS 2 for the Detection of Hemoglobin Variants Based on Their Migration Positions.

In this report, we evaluated utility of the capillary electrophoresis (CE) migration position of the CAPILLARYS 2 CE instrument. The precision of this x-axis number was determined on a selection of common hemoglobin (Hb) variants (Hb S, Hb C, Hb D-Punjab, Hb E, Hb Hope), and the reproducibility of this number was evaluated by comparing the results obtained by two large reference laboratories on 81 Hb variants. Additionally, the CE migration position is given for a total of 409 Hb variants. The x-axis migration position showed excellent intra- and interassay precision. Comparison of Hb variants seen by both laboratories showed that 83% had a difference in migration position of 1 unit or less. Only three rare Hb variants showed a difference of more than 2 units. In summary, the CE migration position is a reproducible value and can be used as an aid in the identification of Hb variants.

DNA interaction studies and photoinduced ligand exchange kinetics of a sterically strained ruthenium(II) complex

Sterically strained ruthenium polypyridyl complexes represent an emerging class of bioactive molecules, capable of photoinduced ligand exchange and covalent binding with DNA, in a manner similar to cisplatin. A sterically strained complex, [Ru(biq)2(dpp)](PF6)2, where biq=2,2′-biquinoline, dpp=2,3-bis(2-pyridyl)pyrazine, was synthesized, and investigated with respect to its photoinduced ligand exchange and DNA interaction properties. Although synthesis and redox properties of the complex, [Ru(biq)2(dpp)](PF6)2, have been reported, the photoinduced ligand exchange properties and DNA binding properties have not yet been reported. Photoinduced dissociation of the dpp ligand was observed upon irradiation of water or acetonitrile solutions of the metal complex with visible light (λirr⩾550nm) to generate [Ru(biq)2(H2O)2](PF6)2 and [Ru(biq)2(CH3CN)2](PF6)2 complexes, respectively. The half-lives of photoactivated exchange of the dpp ligand with water and acetonitrile were found to be 14min and 6min, respectively. The dissociation of the dpp ligand upon irradiation with light was confirmed by mass spectrometry. The synthesized complex displays covalent binding with DNA upon irradiation with visible light, confirmed by slowing down of the rate of DNA migration in agarose gel electrophoresis. No effect on the rate of migration of DNA was observed when incubated with metal complex in dark. The spectroscopic, photoinduced ligand exchange kinetics and DNA interaction properties are presented.

Characterization and pathogenicity of a novel mammalian orthoreovirus from wild short-nosed fruit bats

Mammalian orthoreoviruses (MRVs) have a wide range of geographic distribution and have been isolated from humans and various animals. This study describes the isolation, molecular characterization and analysis of pathogenicity of MRV variant B/03 from wild short-nosed fruit bats. Negative stain electron microscopy illustrated that the B/03 strain is a non-enveloped icosahedral virus with a diameter of 70nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) migration patterns showed that the B/03 viral genome contains 10 segments in a 3:3:4 arrangement. The isolate belongs to MRV serotype 1 based on S1 gene nucleotide sequence data. BALB/c mice experimentally infected with B/03 virus by intranasal inoculation developed severe respiratory distress with tissue damage and inflammation. Lastly, B/03 virus has an increased transmission risk between bats and humans or animals.

Impact of capillary conditioning and background electrolyte composition on capillary electrophoresis analysis of prostate specific antigen isoforms

Glycoproteins expressed in the human body can experience modifications as result of pathological situations. Detection of those changes can be useful as disease biomarkers. As a result of these modifications, size and/or electrical charge of the glycoprotein can be altered. Migration in capillary zone electrophoresis (CZE) is governed by the size to charge ratio of the analyte and therefore this separation technique can be used to monitor those modifications. At its turn, the alteration of the electrophoretical pattern of a given glycoprotein could be used as disease biomarker. To this aim, high repeatability for separation of a large number of peaks for a given glycoprotein is desirable.For prostate cancer, new markers are needed to decrease the high number of false positive results provided by the biomarkers currently used in clinics. In this sense, CZE methods for analysis of the several prostate specific antigen (PSA) peaks which this glycoprotein exhibit, called isoforms and containing one or more glycoforms, could be useful to study the PSA pattern as prostate cancer marker.In this study two complementary strategies to achieve both lot-to-lot capillary repeatability and high resolution of a large number of PSA isoforms are developed. Better performance and precision have been obtained for capillaries conditioned with HCl than for those conditioned with NaOH. Optimization of the background electrolyte (BGE) pH value to 8.0 and inclusion of 3M urea on its composition were the two factors of highest impact for enhancing resolution of the highest number of PSA peaks. Under the optimized conditions for capillary conditioning and BGE pH and composition, long-term resolution of 10 isoforms of PSA was achieved. Inter-day (n=3) %RSD was 0.55 for the ratio tm/tEOF, 1.15 for μeff, and 5.02 for % Acorr of the PSA peaks.

A comparison of gold nanoparticle surface co-functionalization approaches using Polyethylene Glycol (PEG) and the effect on stability, non-specific protein adsorption and internalization

To create clinically useful gold nanoparticle (AuNP) based cancer therapeutics it is necessary to co-functionalize the AuNP surface with a range of moieties; e.g. Polyethylene Glycol (PEG), peptides and drugs. AuNPs can be functionalized by creating either a mixed monolayer by attaching all the moieties directly to the surface using thiol chemistry, or by binding groups to the surface by means of a bifunctional polyethylene glycol (PEG) linker. The linker methodology has the potential to enhance bioavailability and the amount of functional agent that can be attached. While there is a large body of published work using both surface arrangements independently, the impact of attachment methodology on stability, non-specific protein adsorption and cellular uptake is not well understood, with no published studies directly comparing the two most frequently employed approaches. This paper compares the two methodologies by synthesizing and characterizing PEG and Receptor Mediated Endocytosis (RME) peptide co-functionalized AuNPs prepared using both the mixed monolayer and linker approaches. Successful attachment of both PEG and RME peptide using the two methods was confirmed using Dynamic Light Scattering, Fourier Transform Infrared Spectroscopy and gel electrophoresis. It was observed that while the ‘as synthesized’ citrate capped AuNPs agglomerated under physiological salt conditions, all the mixed monolayer and PEG linker capped samples remained stable at 1M NaCl, and were stable in PBS over extended periods. While it was noted that both functionalization methods inhibited non-specific protein attachment, the mixed monolayer samples did show some changes in gel electrophoresis migration profile after incubation with fetal calf serum. PEG renders the AuNP stable in-vivo however, studies with MDA‐MB‐231 and MCF 10A cell lines indicated that functionalization with PEG, blocks cellular uptake. It was observed that co-functionalization with RME peptide using both the mixed monolayer and PEG linker methods greatly enhanced cellular internalization compared to PEG capped AuNPs.

The Gene Transfer Agent RcGTA Contains Head Spikes Needed for Binding to the Rhodobacter capsulatus Polysaccharide Cell Capsule

Viruses and bacteriophages recognize cell surface proteins using receptor-binding proteins. In most tailed bacteriophages, receptor-binding proteins are located on the bacteriophage tail. The gene transfer agent of Rhodobacter capsulatus, RcGTA, morphologically resembles a tailed bacteriophage and binds to a capsular polysaccharide covering R. capsulatus cells. Here, we report that the RcGTA capsid (head) is decorated by spikes that are needed for binding to the capsule. The triangular spikes measured ~12nm and appeared to be attached at the capsid vertices. Head spike production required the putative carbohydrate-binding protein ghsB (rcc01080) previously thought to encode a side tail fiber protein. We found that ghsB is likely co-transcribed with ghsA (rcc01079) and that ghsA/ghsB is regulated by the CckA-ChpT-CtrA phosphorelay homologues and a quorum-sensing system. GhsA and GhsB were found to be CckA-dependent RcGTA maturation factors, as GhsA- and GhsB-deficient particles were found to have altered native-gel electrophoresis migration. Additionally, we provide electron microscopy images showing that RcGTA contains side tail fibers and a baseplate-like structure near the tip of the tail, which are independent of ghsB.