Phase and fluorescence imaging with a surprisingly simple microscope based on chromatic aberration.

We propose a simple and compact microscope combining phase imaging with multi-color fluorescence using a standard bright-field objective. The phase image of the sample is reconstructed from a single, approximately 100 µm out-of-focus image taken under semi-coherent illumination, while fluorescence is recorded in-focus in epi-fluorescence geometry. The reproducible changes of the focus are achieved with specifically introduced chromatic aberration in the imaging system. This allows us to move the focal plane simply by changing the imaging wavelength. No mechanical movement of neither sample nor objective or any other part of the setup is therefore required to alternate between the imaging modality. Due to its small size and the absence of motorized components the microscope can easily be used inside a standard biological incubator and allows long-term imaging of cell culture in physiological conditions. A field-of-view of 1.2 mm2 allows simultaneous observation of thousands of cells with micro-meter spatial resolution in phase and multi-channel fluorescence mode. In this manuscript we characterize the system and show a time-lapse of cell culture in phase and multi-channel fluorescence recorded inside an incubator. We believe that the small dimensions, easy usage and low cost of the system make it a useful tool for biological research.

Extracellular DNA Provides Structural Integrity to a Micrococcus luteus Biofilm.

Force spectroscopy was used to show that extracellular DNA (eDNA) has a pre-eminent structural role in a biofilm. The adhesive behavior of extracellular polymeric substances to poly(ethylene terephthalate), a model hydrophobic surface, was measured in response to their degradation by hydrolytic enzymes known for their biofilm dispersion potential: DNaseI, protease, cellulase, and mannanase. Only treatment with DNaseI significantly decreased the adhesive force of the model bacterium Micrococcus luteus with the surface, and furthermore this treatment almost completely eliminated any components of the biofilm maintaining the adhesion, establishing a key structural role for eDNA.

High-Temperature Spray-Dried Polymer/Bacteria Microparticles for Electrospinning of Composite Nonwovens.

Living Micrococcus luteus (M. luteus) and Escherichia coli (E. coli) are encapsulated in poly(vinyl alcohol), poly(vinylpyrrolidone), hydroxypropyl cellulose, and gelatin by high-temperature spray drying. The challenge is the survival of the bacteria during the standard spray-drying process at temperatures of 150 °C (M. luteus) and 120 °C (E. coli). Raman imaging and transmission electron microscopy indicate encapsulated bacteria in hollow composite microparticles. The versatility of the spray-dried polymer bacteria microparticles is successfully proved by standard polymer solution-processing techniques such as electrospinning, even with harmful solvents, to water-insoluble polyacrylonitrile, polystyrene, poly(methyl methacrylate), and poly(vinyl butyrate) nanofiber nonwovens, which opens numerous new opportunities for novel applications.

The bacteriolytic activity of native and covalently immobilized lysozyme against Gram-positive and Gram-negative bacteria is differentially affected by charged amino acids and glycine.

The emergence of new antibiotic-resistant bacterial strains means it is increasingly important to find alternatives to traditional antibiotics, such as bacteriolytic enzymes. The bacteriolytic enzyme lysozyme is widely used in medicine as an antimicrobial agent, and covalent immobilization of lysozyme can expand its range of possible applications. However, information on the effect of such immobilized preparations on whole bacterial cells is quite limited. Here, we demonstrate the differential effects of glycine and charged (basic and acidic) amino acids on the enzymatic lysis of Gram-positive and Gram-negative bacteria by soluble and immobilized lysozyme. Glycine and basic amino acids (histidine, lysine, and arginine) significantly increase the rate of lysis of Gram-negative Escherichia coli cells in the presence of soluble lysozyme, but they do not substantially affect the rate of enzymatic lysis of Gram-positive Micrococcus luteus. Glutamate and aspartate significantly enhance enzymatic lysis of both E. coli and M. luteus. When using immobilized lysozyme, the effects of amino acids on the rate of cell lysis are significantly reduced. For immobilized lysozyme, the presence of an external diffusion mode on cell lysis kinetics at bacterial concentrations below 4 × 108 colony-forming units·mL-1 was shown. The broadening of the pH optimum of lysozyme activity after immobilization has been demonstrated for both Gram-positive and Gram-negative bacteria. The Michaelis constant (Km) values of immobilized lysozyme were increased by 1.5-fold for E. coli cell lysis and 4.6-fold for M. luteus cell lysis compared to soluble enzyme. A greater understanding of the effect of amino acids on the activity of native and immobilized lysozyme is important for both the development of new materials for medical purposes and elucidating the interaction of lysozyme with bacterial cells. Of particular interest is our finding that lysozyme activity against Gram-negative bacteria is enhanced in the presence of glycine and charged amino acids over a wide range of concentrations.

The disruptive effect of lysozyme on the bacterial cell wall explored by an in-silico structural outlook.

The bacterial cell wall, a structural unit of peptidoglycan polymer comprised of glycan strands consisting of a repeating disaccharide motif [N-acetylglucosamine (NAG) and N-acetylmuramylpentapeptide (NAM pentapeptide)], encases bacteria and provides structural integrity and protection. Lysozymes are enzymes that break down the bacterial cell wall and disrupt the bacterial life cycle by cleaving the linkage between the NAG and NAM carbohydrates. Lab exercises focused on the effects of lysozyme on the bacterial cell wall are frequently incorporated in biochemistry classes designed for undergraduate students in diverse fields as biology, microbiology, chemistry, agronomy, medicine, and veterinary medicine. Such exercises typically do not include structural data. We describe here a sequence of computer tasks designed to illustrate and reinforce both physiological and structural concepts involved in lysozyme effects on the bacterial cell-wall structure. This lab class usually lasts 3.5 hours. First, the instructor presents introductory concepts of the bacterial cell wall and the effect of lysozyme on its structure. Then, students are taught to use computer modeling to visualize the three-dimensional structure of a lysozyme in complex with bacterial cell-wall fragments. Finally, the lysozyme inhibitory effect on a bacterial culture is optionally proposed as a simple microbiological assay. The computer lab exercises described here give students a realistic understanding of the disruptive effect of lysozymes on the bacterial cell wall, a crucial component in bacterial survival. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(1):83-90, 2018.

Antibacterial active compounds from Hypericum ascyron L. induce bacterial cell death through apoptosis pathway.

Hypericum ascyron L. has been used as a traditional medicine for the treatment of wounds, swelling, headache, nausea and abscesses in China for thousands of years. However, modern pharmacological studies are still necessary to provide a scientific basis to substantiate their traditional use. In this study, the mechanism underlying the antimicrobial effect of the antibacterial activity compounds from H. ascyron L. was investigated. Bioguided fractionation of the extract from H. ascyron L. afforded antibacterial activity fraction 8. The results of cup plate analysis and MTT assay showed that the MIC and MBC of fraction 8 is 5 mg/mL. Furthermore, using Annexin V-FITC/PI, TUNEL labeling and DNA gel electrophoresis, we found that cell death with apoptosis features similar to those in eucaryon could be induced in bacteria strains after exposure to the antibacterial activity compounds from H. ascyron L. at moderate concentration. In addition, we further found fraction 8 could disrupt the cell membrane potential indicate that fraction 8 exerts pro-apoptotic effects through a membrane-mediated apoptosis pathway. Finally, quercetin and kaempferol 3-O-ß-(2″-acetyl)-galactopyranoside, were identified from fraction 8 by means of Mass spectrometry and Nuclear magnetic resonance. To our best knowledge, this study is the first to show that Kaempferol 3-O-ß-(2″-acetyl)-galactopyranoside coupled with quercetin had significant antibacterial activity via apoptosis pathway, and it is also the first report that Kaempferol 3-O-ß-(2″-acetyl)-galactopyranoside was found in clusiacea. Our data might provide a rational base for the use of H. ascyron L. in clinical, and throw light on the development of novel antibacterial drugs.

Biosorption of lead and copper by heavy-metal tolerant Micrococcus luteus DE2008.

Micrococcus luteus DE2008 has the ability to absorb lead and copper. The effect of these metals on biomass and viability of this microorganism were investigated and removal of the metals from culture media was determined. Lead had no effect on the biomass expressed as mg Carbon/cm(3) of M. Iuteus DE2008, but in the case of copper, the minimum metal concentration that affected the biomass was 0.1 mM Cu(II). According to these results this microorganism shows a greater tolerance for lead. The minimum metal concentration that affected viability (expressed as the percentage of live cells) was 0.5 mM for both metals. M. luteus DE2008 exhibited a specific removal capacity of 408 mg/g for copper and 1965 mg/g for lead. This microorganism has a greater ability to absorb Pb(II) than Cu(II). M. luteus DE2008 could be seen as a microorganism capable of restoring environments polluted by lead and copper.

A lysozyme and magnetic bead based method of separating intact bacteria.

As a response to environmental stress, bacterial cells can enter a physiological state called viable but noncultivable (VBNC). In this state, bacteria fail to grow on routine bacteriological media. Consequently, standard methods of contamination detection based on bacteria cultivation fail. Although they are not growing, the cells are still alive and are able to reactivate their metabolism. The VBNC state and low bacterial densities are big challenges for cultivation-based pathogen detection in drinking water and the food industry, for example. In this context, a new molecular-biological separation method for bacteria using point-mutated lysozymes immobilised on magnetic beads for separating bacteria is described. The immobilised mutated lysozymes on magnetic beads serve as bait for the specific capture of bacteria from complex matrices or water due to their remaining affinity for bacterial cell wall components. Beads with bacteria can be separated using magnetic racks. To avoid bacterial cell lysis by the lysozymes, the protein was mutated at amino acid position 35, leading to the exchange of the catalytic glutamate for alanine (LysE35A) and glutamine (LysE35Q). As proved by turbidity assay with reference bacteria, the muramidase activity was knocked out. The mutated constructs were expressed by the yeast Pichia pastoris and secreted into expression medium. Protein enrichment and purification were carried out by SO(3)-functionalised nanoscale cationic exchanger particles. For a proof of principle, the proteins were biotinylated and immobilised on streptavidin-functionalised, fluorescence dye-labelled magnetic beads. These constructs were used for the successful capture of Syto9-marked Microccocus luteus cells from cell suspension, as visualised by fluorescence microscopy, which confirmed the success of the strategy.

Polymer/bacteria composite nanofiber non-wovens by electrospinning of living bacteria protected by hydrogel microparticles.

Physically crosslinked PVA-hydrogel microparticles are utilized for encapsulation of E. coli and M. luteus. The bacteria survive dry storage or treatment with bacteria-hostile organic solvents significantly better than unprotected bacteria as proven by culture-test experiments. The bacteria-protecting PVA microparticles are available for standard polymer-solution-processing techniques, as exemplarily shown by co-electrospinning of living bacteria encapsulated in dry PVA-hydrogel microparticles together with PVB-, PLLA-, and PCL-form organic solvents.

Effect of enzyme modification by well-defined multi-armed poly(ethylene glycol) synthesized using polyamidoamine dendron.

Egg white lysozyme was chemically modified by PEGylated PAMAM 1st, 2nd and 3rd generation dendrons, which had 2, 4 and 8 PEG arms, respectively. The number of PEG chains introduced to the lysozyme molecule drastically increased with an increase in dendron generation, although the number of PEGylated PAMAM dendrons introduced decreased due to steric repulsion. The lytic activity of lysozyme to Micrococcus luteus cells was effectively inhibited by conjugating PEGylated PAMAM dendron to the lysozyme, indicating steric stabilization of PEG chains at the surface of lysozyme molecule. In addition, the enzymatic reaction of the lysozyme with oligosaccharide substrate was apparently accelerated by a substrate condensation effect due to the multi-armed structure of PEG.

19F NMR analysis of the antimicrobial peptide PGLa bound to native cell membranes from bacterial protoplasts and human erythrocytes.

(19)F NMR is a unique tool to examine the structure of fluorine-labeled peptides in their native cellular environment, due to an exquisite sensitivity and lack of natural abundance background. For solid-state NMR analysis, we isolated native membranes from erythrocyte ghosts and bacterial protoplasts and prepared them as macroscopically oriented samples. They showed a high purity and quality of alignment according to (31)P NMR, and the membrane-bound antimicrobial peptide PGLa could be detected by (19)F NMR. The characteristic fingerprint splitting of its (19)F reporter group indicated that the peptide helix binds to the native membranes in a surface alignment, albeit with a higher affinity in the prokaryotic than the eukaryotic system.

Purification and characterization of enterocin LR/6, a bacteriocin from Enterococcus faecium LR/6.

Enterocin LR/6, a bacteriocin obtained from the culture filtrate of Enterococcus faecium strain LR/6, has been purified to homogeneity using ammonium sulfate precipitation, cation-exchange chromatography, gel-filtration, and checked on reverse-phase high-performance liquid chromatography. It is active at high temperatures (boiling as well as autoclaving) and over a wide range of pH (2.0-8.0). Also, it is sensitive to a number of proteolytic enzymes but is stable in the presence of surfactants and organic solvents. The protein could be stored at least up to 1 year at low temperatures (4 degrees C and -20 degrees C) without any loss of activity. The N-terminal sequence of enterocin LR/6 showed no homology with known enterocins or other bacteriocins present in the database, suggesting it to be a novel enterocin. Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed its mass to be approximately 6.1 kDa. It showed a bactericidal mode of action against indicator strain, Micrococcus luteus.

The antibacterial and antifungal activity of a soda-lime glass containing silver nanoparticles.

The antibacterial and antifungal activity of a low melting point soda-lime glass powder containing silver nanoparticles has been studied. Nano-Ag sepiolite fibres containing monodispersed silver nanoparticles (d(50) approximately 11 +/- 9 nm) were used as the source of silver. This powder presents a high antibacterial (against gram-positive and gram-negative bacteria) as well as antifungal (against I. orientalis) activity. The observed high activity against yeast has been explained by considering the inhibitory effect of the Ca(2+) lixiviated from the glass on the growth of the yeast colonies.

Recombinant expression of human cathelicidin (hCAP18/LL-37) in Pichia pastoris.

The constitutive expression of human cathelicidin LL-37 antimicrobial peptide was achieved using the methylotrophic yeast, Pichia pastoris. An LL-37 cDNA clone was amplified by PCR using human fetal cDNA library as template. The 111 bp fragment encoding mature LL-37 gene was subcloned into pGAPZ-E, an episomal form of the pGAPZB vector incorporating PARS1. It was then transformed into the P. pastoris X-33 strain for intracellular expression. A small peptide with a molecular mass of about 5 kDa was detected by 17% peptide-PAGE analysis. The recombinant LL-37 peptide was purified from the gel and its amino acid sequence was determined by LC-ESI-MS/MS analysis. The initiating amino acid, methionine, was still attached to the N-terminal region of recombinant LL-37. LL-37 crude extract from P. pastoris showed an antimicrobial activity against Micrococcus luteus as the test strain. The successful expression of human LL-37 indicates that the system may be applicable to the expression of other human defensins without resorting to fusion protein constructions.

Proteins of the Rpf (resuscitation promoting factor) family are peptidoglycan hydrolases.

The secreted Micrococcus luteus protein, Rpf, is required for successful resuscitation of dormant "non-culturable" M. luteus cells and for growth stimulation in poor media. The biochemical mechanism of Rpf action remained unknown. Theoretical predictions of Rpf domain architecture and organization, together with a recent NMR analysis of the protein structure, indicate that the conserved Rpf domain has a lysozyme-like fold. In the present study, we found that both the secreted native protein and the recombinant protein lyse crude preparations of M. luteus cell walls. They also hydrolyze 4-methylumbelliferyl-beta-D-N,N',N''-triacetylchitotrioside, a synthetic substrate for peptidoglycan muramidases, with optimum activity at pH 6. The Rpf protein also has weak proteolytic activity against N-CBZ-Gly-Gly-Arg-beta-naphthylamide, a substrate for trypsin-like enzymes. Rpf activity towards 4-methylumbelliferyl-beta-D-N,N',N''-triacetylchitotrioside was reduced when the glutamate residue at position 54, invariant for all Rpf family proteins and presumably involved in catalysis, was altered. The same amino acid substitution resulted in impaired resuscitation activity of Rpf. The data indicate that Rpf is a peptidoglycan-hydrolyzing enzyme, and strongly suggest that this specific activity is responsible for its growth promotion and resuscitation activity. A possible mechanism of Rpf-mediated resuscitation is discussed.

[Cell aggregation in cultures of Micrococcus luteus studied by dynamic light scattering].

Cell aggregation was studied using the method of dynamic light scattering in the course of growth of Micrococcus luteus cultures in a liquid medium. The method detects particles ranging in size from 0.5 to 1000 microm in samples containing no more than 10(5) cells/ml. When grown in liquid media, M. luteus forms aggregates; during the lag phase, 80% of the cells are found in aggregates of 10 to 1000 microm, only minor amounts being represented by single cells. With the onset of exponential growth, the aggregates were decomposed, and single cells became prevalent in the culture liquid. This observation confirms that the aggregation of the cells during the lag phase is prerequisite to the initiation of bacterial growth. The method may be used in biotechnology for monitoring the state of bacterial cultures.

Construction of biofilms with defined internal architecture using dielectrophoresis and flocculation.

A novel approach was developed for the construction of biofilms with defined internal architecture using AC electrokinetics and flocculation. Artificial structured microbial consortia (ASMC) consisting of localized layered microcolonies of different cell types were formed by sequentially attracting different cell types to high field regions near microelectrodes using dielectrophoresis. Stabilization of the microbial consortia on the electrode surface was achieved by crosslinking the cells using the flocculant polyethyleneimine (PEI). Consortia of Escherichia coli, Micrococcus luteus, and Saccharomyces cerevisiae were made as model systems. Also, more natural consortia were made of the bacteria Pseudomonas putida, Clavibacter michiganense, and Methylobacterium mesophilum, which are found together in consortia during biodegradation of metal-cutting waste fluids.

[Lysozyme Reference Standard (Control 031) of National Institute of Health Sciences].

The "Lysozyme Reference Standard (Control 951031)" of the National Institute of Health Sciences was prepared. The lysozyme potency of the standard material was assayed against the Lysozyme Reference Standard (Control 951) by turbidimetric method two turbidimetric methods using the dried y-cells of Micrococcus luteus as a substrate. The potency of the standard material was in satisfactory agreement with that of Lysozyme Reference Standard (Control 951) and was defined as 1 mg [potency] per mg.

Fluorescent brighteners: novel stains for the flow cytometric analysis of microorganisms.

Flow cytometry is a rapid method for measuring the optical properties of individual cells. The technique has found great utility in the study of mammalian cells, but microbiological applications have been more limited. We here show that UV-excited fluorescent whitening agents, in particular Tinopal CBS-X, are effective stains for both vegetative microbial cells and for spores of Gram-positive bacteria. Pretreatment of samples with ethanol speeds the staining process. Under favourable conditions, Tinopal CBS-X may be used to discriminate among organisms, a fact that may be useful when screening for a target microorganism against a high biological background.