Disruption of Bacterial Cells by a Synthetic Zeolite

The use of a synthetic zeolite (type 4A, Union Carbide Corp., Linde Div., New York, N.Y.) in a procedure for the preparation of pure cell wall fractions proved successful for many gram-positive, gram-negative, and acid-fast bacteria, as well as for some fungi. The technique, however, was found to be limited in effectiveness for Rhodospirillum rubrum, Gaffkya tetragena, and Sarcina lutea, and not applicable to preparations of heat killed microorganisms. The possible mechanisms of zeolite action, together with the effect of the disruptive procedure on the chemical composition of cell wall fragments, were investigated also.     

Autonomous Synchronization of Chemically Coupled Synthetic Oscillators

Synthetic biology has recently provided functional single-cell oscillators. With a few exceptions, however, synchronization across a population has not been achieved yet. In particular, designing a cell coupling mechanism to achieve autonomous synchronization is not straightforward since there are usually several different design alternatives. Here, we propose a method to mathematically predict autonomous synchronization properties, and to identify the network structure with the best performance, thus increasing the feasibility for a successful implementation in vivo.     

Catecholamine Secretion by Chemically Skinned Cultured Chromaffm Cells

The secretory system of intact chromaffin cells is not accessible to direct chemical manipulation because of the selective permeability of the plasmalemma. We have devised a simple procedure for chemically "skinning" (permeabilizing) cultured adrenal medullary chromaffin cells by brief exposure to the detergent saponin. This procedure disrupts the continuity of the plasmalemma, thus allowing us to bypass those aspects of the secretory process controlled by the cell membrane and giving direct access to exogenous substances to the cellular secretory machinery. We report here that the skinned cells retain a fully competent secretory mechanism dependent only on exogenous calcium and MgATP. Saponin treatment had no significant effect on the total catecholamine content of the cells. Secretion could be initiated by either MgATP or calcium as long as the other was present in the medium. Catecholamine and dopamine-beta-hydroxylase release by the skinned cells was dependent on the calcium concentration of the medium. The ratio of secreted catecholamine and enzyme was similar to that of the cells, indicating that secretion occurred by an exocytotic mechanism. About half the total cellular content of the cytoplasmic enzyme lactic dehydrogenase was released during the permeabilization process and subsequent incubations, indicating plasmalemma permeability to molecules as large as protein. Calcium-induced secretion was unaffected by several drugs known to affect catecholamines and granule function. Saponin treatment of chromaffin cells in culture appears to be a simple means for allowing access to exogenous substances to the cells' secretory machinery. Therefore, it offers the opportunity to use chemical treatments, and perhaps specific antibodies to cellular components, to determine the role of these elements in the secretory process. These techniques should also be applicable to other cells known to secrete by an exocytotic mechanism.     

Synthetic Surface for Expansion of Human Mesenchymal Stem Cells in Xeno-Free,Chemically Defined Culture Conditions

Human mesenchymal stem cells (hMSCs) possess three properties of great interest for the development of cell therapies and tissue engineering: multilineage differentiation, immunomodulation, and production of trophic factors. Efficient ex vivo expansion of hMSCs is a challenging requirement for large scale production of clinical grade cells. Low-cost, robust, scalable culture methods using chemically defined materials need to be developed to address this need. This study describes the use of a xeno-free synthetic peptide acrylate surface, the Corning® Synthemax® Surface, for culture of hMSCs in serum-free, defined medium. Cell performance on the Corning Synthemax Surface was compared to cells cultured on biological extracellular matrix (ECM) coatings in xeno-free defined medium and in traditional conditions on tissue culture treated (TCT) plastic in fetal bovine serum (FBS) supplemented medium. Our results show successful maintenance of hMSCs on Corning Synthemax Surface for eight passages, with cell expansion rate comparable to cells cultured on ECM and significantly higher than for cells in TCT/FBS condition. Importantly, on the Corning Synthemax Surface, cells maintained elongated, spindle-like morphology, typical hMSC marker profile and in vitro multilineage differentiation potential. We believe the Corning Synthemax Surface, in combination with defined media, provides a complete synthetic, xeno-free, cell culture system for scalable production of hMSCs.     

Substrate-Specific Development of Thermophilic Bacterial Consortia by Using Chemically Pretreated Switchgrass

Microbial communities that deconstruct plant biomass have broad relevance in biofuel production and global carbon cycling. Biomass pretreatments reduce plant biomass recalcitrance for increased efficiency of enzymatic hydrolysis. We exploited these chemical pretreatments to study how thermophilic bacterial consortia adapt to deconstruct switchgrass (SG) biomass of various compositions. Microbial communities were adapted to untreated, ammonium fiber expansion (AFEX)-pretreated, and ionic-liquid (IL)-pretreated SG under aerobic, thermophilic conditions using green waste compost as the inoculum to study biomass deconstruction by microbial consortia. After microbial cultivation, gravimetric analysis of the residual biomass demonstrated that both AFEX and IL pretreatment enhanced the deconstruction of the SG biomass approximately 2-fold. Two-dimensional nuclear magnetic resonance (2D-NMR) experiments and acetyl bromide-reactive-lignin analysis indicated that polysaccharide hydrolysis was the dominant process occurring during microbial biomass deconstruction, and lignin remaining in the residual biomass was largely unmodified. Small-subunit (SSU) rRNA gene amplicon libraries revealed that although the dominant taxa across these chemical pretreatments were consistently represented by members of the Firmicutes, the Bacteroidetes, and Deinococcus-Thermus, the abundance of selected operational taxonomic units (OTUs) varied, suggesting adaptations to the different substrates. Combining the observations of differences in the community structure and the chemical and physical structure of the biomass, we hypothesize specific roles for individual community members in biomass deconstruction.     

Tanzawaic Acids,a Chemically Novel Set of Bacterial Conjugation Inhibitors

Bacterial conjugation is the main mechanism for the dissemination of multiple antibiotic resistance in human pathogens. This dissemination could be controlled by molecules that interfere with the conjugation process. A search for conjugation inhibitors among a collection of 1,632 natural compounds, identified tanzawaic acids A and B as best hits. They specially inhibited IncW and IncFII conjugative systems, including plasmids mobilized by them. Plasmids belonging to IncFI, IncI, IncL/M, IncX and IncH incompatibility groups were targeted to a lesser extent, whereas IncN and IncP plasmids were unaffected. Tanzawaic acids showed reduced toxicity in bacterial, fungal or human cells, when compared to synthetic conjugation inhibitors, opening the possibility of their deployment in complex environments, including natural settings relevant for antibiotic resistance dissemination.     

Ballistic Disruption of Penicillium chrysogenum Cells

The use of glass beads in a high-speed mixing device to rupture organisms was applied to molds. The use of a mixer in which the propellor shaft enters from the top into a metal mixing chamber made it possible to immerse the whole device in a salt water and ice mixture so that the temperature of the glass-bead slurry could be kept below 5 C without difficulty. Mycelia, glass beads, and buffer in a 1:2:3 (w/w) ratio gave above 95% breakage in 15 min with Penicillium chrysogenum cells and in 4 min with Rhizopus nigricans. Some of the factors influencing breakage are discussed.     

Quantitative Determination of Dye Uptake by Bacterial Cells

A colorimetric method is presented for the quantitative determination of dye uptake by bacterial cells. Experiments showed that the dye to cell ratio was of major importance in controlling the amount of dye taken up per weight of bacterial cells. Approximate dye saturation of cells could be obtained (at pH 6.1 to 6.3) although the dye uptake curves did not absolutely level off.     

Rapid and Simple Quantification of Bacterial Cells by Using a Microfluidic Device

This study investigated a microfluidic chip-based system (on-chip flow cytometry) for quantification of bacteria both in culture and in environmental samples. Bacterial numbers determined by this technique were similar to those obtained by direct microscopic count. The time required for this on-chip flow cytometry was only 30 min per 6 samples.     

Disruption of Bacterial Cell-to-Cell Communication by Marine Organisms and its Relevance to Aquaculture

Bacterial disease is one of the most critical problems in commercial aquaculture. Although various methods and treatments have been developed to curb the problem, yet they still have significant drawbacks. A novel and environmental-friendly approach in solving this problem is through the disruption of bacterial communication or quorum sensing (QS). In this communication scheme, bacteria regulate their own gene expression by producing, releasing, and sensing chemical signals from the environment. There seems to be a link between QS and diseases through the regulation of certain phenotypes and the induction of virulence factors responsible for pathogen–host association. Several findings have reported that numerous aquatic organisms such as micro-algae, macro-algae, invertebrates, or even other bacteria have the potential to disrupt QS. The mechanism of action varies from degradation of signals through enzymatic or chemical inactivation to antagonistic as well as agonistic activities. This review focuses on the existing marine organisms that are able to interfere with QS with potential application for aquaculture as bacterial control.     

Breakdown of Diazotized Proteins and Synthetic Substrates by Rumen Bacterial Proteases

Several different kinds of substrate were used to investigate the proteolytic activity of rumen bacteria and of proteases released from rumen bacteria by blending (“coat proteases”). These substrates included diazotized feed proteins and diazotized soluble and insoluble pure proteins. It was concluded that, while solubility was an important factor, the secondary and tertiary structure of a protein had a major influence on its rate of digestion. The resistance of elastin congo red to digestion indicated that similar fibrous proteins in plant material might resist proteolytic attack by rumen bacteria. Coat proteases had a broad specificity, including several exo- and endopeptidase activities, as determined by using synthetic peptide substrates.     

Chemical Communication of Antibiotic Resistance by a Highly Resistant Subpopulation of Bacterial Cells

The overall antibiotic resistance of a bacterial population results from the combination of a wide range of susceptibilities displayed by subsets of bacterial cells. Bacterial heteroresistance to antibiotics has been documented for several opportunistic Gram-negative bacteria, but the mechanism of heteroresistance is unclear. We use Burkholderia cenocepacia as a model opportunistic bacterium to investigate the implications of heterogeneity in the response to the antimicrobial peptide polymyxin B (PmB) and also other bactericidal antibiotics. Here, we report that B. cenocepacia is heteroresistant to PmB. Population analysis profiling also identified B. cenocepacia subpopulations arising from a seemingly homogenous culture that are resistant to higher levels of polymyxin B than the rest of the cells in the culture, and can protect the more sensitive cells from killing, as well as sensitive bacteria from other species, such as Pseudomonas aeruginosa and Escherichia coli. Communication of resistance depended on upregulation of putrescine synthesis and YceI, a widely conserved low-molecular weight secreted protein. Deletion of genes for the synthesis of putrescine and YceI abrogate protection, while pharmacologic inhibition of putrescine synthesis reduced resistance to polymyxin B. Polyamines and YceI were also required for heteroresistance of B. cenocepacia to various bactericidal antibiotics. We propose that putrescine and YceI resemble "danger" infochemicals whose increased production by a bacterial subpopulation, becoming more resistant to bactericidal antibiotics, communicates higher level of resistance to more sensitive members of the population of the same or different species.     

Disruption of Type I Interferon Induction by HIV Infection of T Cells

Our main objective of this study was to determine how Human Immunodeficiency Virus (HIV) avoids induction of the antiviral Type I Interferon (IFN) system. To limit viral infection, the innate immune system produces important antiviral cytokines such as the IFN. IFN set up a critical roadblock to virus infection by limiting further replication of a virus. Usually, IFN production is induced by the recognition of viral nucleic acids by innate immune receptors and subsequent downstream signaling. However, the importance of IFN in the defense against viruses has lead most pathogenic viruses to evolve strategies to inhibit host IFN induction or responses allowing for increased pathogenicity and persistence of the virus. While the adaptive immune responses to HIV infection have been extensively studied, less is known about the balance between induction and inhibition of innate immune defenses, including the antiviral IFN response, by HIV infection. Here we show that HIV infection of T cells does not induce significant IFN production even IFN I Interferon production. To explain this paradox, we screened HIV proteins and found that two HIV encoded proteins, Vpu and Nef, strongly antagonize IFN induction, with expression of these proteins leading to loss of expression of the innate immune viral RNA sensing adaptor protein, IPS-1 (IFN-β promoter stimulator-1). We hypothesize that with lower levels of IPS-1 present, infected cells are defective in mounting antiviral responses allowing HIV to replicate without the normal antiviral actions of the host IFN response. Using cell lines as well as primary human derived cells, we show that HIV targeting of IPS-1 is key to limiting IFN induction. These findings describe how HIV infection modulates IFN induction providing insight into the mechanisms by which HIV establishes infection and persistence in a host.     

Monitoring Bacterial Transport by Stable Isotope Enrichment of Cells

Understanding the transport and behavior of bacteria in the environment has broad implications in diverse areas, ranging from agriculture to groundwater quality, risk assessment, and bioremediation. The ability to reliably track and enumerate specific bacterial populations in the context of native communities and environments is key to developing this understanding. We report a novel bacterial tracking approach, based on altering the stable carbon isotope value (δ¹³C) of bacterial cells, which provides specific and sensitive detection and quantification of those cells in environmental samples. This approach was applied to the study of bacterial transport in saturated porous media. The transport of introduced organisms was indicated by mass spectrometric analysis of groundwater samples, where the presence of ¹³C-enriched bacteria resulted in increased δ¹³C values of the samples, allowing specific and sensitive detection and enumeration of the bacteria of interest. We demonstrate the ability to produce highly ¹³C-enriched bacteria, present data indicating that results obtained with this approach accurately represent intact introduced bacteria, and include field data on the use of this stable isotope approach to monitor in situ bacterial transport. This detection strategy allows sensitive detection of an introduced, unmodified bacterial strain in the presence of the indigenous bacterial community, including itself in its unenriched form.     

Effect of Synthetic Hydroxy Isothiocyanates on a Bacterial Virus and DNA

The Effect of hydroxy isothiocyanates on a bacterial virus and M13 DNA was examined. Hydroxy-substituted phenyl and phenyl alkyl isothiocyanates, especially 2-(3,4-dihydroxyphenyl)ethyl isothiocyanate(IT-Dop) synthesized from dopamine, showed antiviral activity on φK. In transfection experiments with M13 mp DNA species, IT-Dop inhibited the single-stranded (SS) molecule more effectively than the double stranded replicative form (RF) DNA. These effects were dependent on reaction time, and on IT-Dop concentration. An additional experiment indicated that treatment with IT-Dop suppressed annealing (reassociation) of denatured DNA. These results indicate that IT-Dop reacts mildly with virus and SS DNA.     

Disruption of EGF Feedback by Intestinal Tumors and Neighboring Cells in Drosophila

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Insecticidal Activity of a Bacterial Crystal Protein Expressed by a Recombinant Baculovirus in Insect Cells

Baculoviruses are insect pathogens with a relatively slow speed of action, and this has limited their use as control agents of insect pests. Introduction into baculoviruses of genes which code for proteins interfering specifically with insect metabolism or metamorphosis, such as toxins, hormones, and enzymes, may enhance the pathogenicity of these viruses. The complete insecticidal crystal protein gene cryIA(b) of Bacillus thuringiensis subsp. aizawai 7.21 was engineered into the nuclear polyhedrosis virus of Autographa californica (AcNPV) in place of the polyhedrin gene. In infected Spodoptera frugiperda cells, the cryIA(b) gene was expressed at a high level without interference with AcNPV production. The crystal protein was found in the cytoplasm of S. frugiperda cells, mainly as large crystals with an ultrastructure similar to that of B. thuringiensis crystals. Infected-cell extracts inhibited feeding of the large cabbage white Pieris brassicae. The toxicity of the crystal protein expressed by AcNPV recombinants was comparable with that of the crystal protein expressed by a corresponding Escherichia coli recombinant.