Isolation and characterization of marine oligotrophic bacteria

A significant part of the world ocean is characterized by low absolute nutrients and chlorophyll concentrations. In these oligotrophic environments, bacteria are very abundant and play a vital role in the remineralization of the dissolved organic matter. Bacteria adapted to oligotrophic waters differ from those adapted to richer environments by some genetic and metabolic characteristics. Culture techniques in bacteriology are based on rich media and do not allow the growth of most marine bacteria. New techniques have been developed for the culture of oligotrophic bacteria, which allow to isolate unknown bacteria. Pelagibacter ubique and Sphingopyxis alaskensis belong to these bacteria recently isolated from the marine environment and their study yielded better understanding of how marine bacteria adapt to oligotrophic conditions.     

In situ rheology of yeast biofilms

The aim of the present work was to investigate the in situ rheological behavior of yeast biofilms growing on stainless steel under static and turbulent flow. The species used (Rhodototula mucilaginosa, Candida krusei, Candida kefyr and Candida tropicalis) were isolated from a clarified apple juice industry. The flow conditions impacted biofilm composition over time, with a predominance of C. krusei under static and turbulent flow. Likewise, structural variations occurred, with a tighter appearance under dynamic flow. Under turbulent flow there was an increase of 112 μm in biofilm thickness at 11 weeks (p < 0.001) and cell morphology was governed by hyphal structures and rounded cells. Using the in situ growth method introduced here, yeast biofilms were determined to be viscoelastic materials with a predominantly solid-like behavior, and neither this nor the G’₀ values were significantly affected by the flow conditions or the growth time, and at large deformations their weak structure collapsed beyond a critical strain of about 1.5–5%. The present work could represent a starting point for developing in situ measurements of yeast rheology and contribute to a thin body of knowledge about fungal biofilm formation.     

In situ pulse respirometric methods for the estimation of kinetic and stoichiometric parameters in aerobic microbial communities

In situ pulse respirometry was applied in an activated sludge bubble column treating synthetic wastewater for the estimation of the (i) maximum specific oxygen consumption rate, (ii) substrate affinity constant, (iii) biomass growth yield, (iv) maintenance coefficient, and (v) specific endogenous respiration rate. Parameters obtained from respirometry were compared to those obtained by the chemostat method, based on substrate and biomass measurements, under several dilution rates. The low sensitivity of substrate measurement methods and the difficulties of sampling heterogeneous biomass suspension are critical issues limiting the applicability of the chemostat method. Additionally, the extensive time consuming nature of this method allows concluding that chemostat method presents several disadvantages in comparison with in situ pulse respirometric techniques. Parameters were obtained from respirograms by fitting ASM1 and ASM3 models, and from experiments performed by injecting pulses of increasing substrate concentration. The injection of pulses of increasing concentration was the most adequate method, with several advantages such as a simpler experimental data interpretation, and results with better confidence.Considering the assessment and comparison of the experimental and calculation methods presented, it is recommended that the estimation of kinetic and stoichiometric parameters in mixed aerobic cultures should preferentially be performed by using in situ respirometric techniques.     

In situ kinetic parameters of glucose-6-phosphatase in the rat liver lobulus.

Glucose-6-phosphatase activity has been determined in periportal and pericentral zones of the rat liver lobule using a quantitative histochemical method. The study was performed on unfixed cryostat sections of livers from fasted and fed female and male rats. Highest activity was found in periportal zones, and starvation caused a 2-3-fold increase of glucose-6-phosphatase activity in periportal and pericentral zones of both sexes. Unexpectedly, KM values were also significantly different in periportal and pericentral zones and were found to increase linearly with Vmax values, irrespective of sex and feeding condition. Because the cryofixation procedure was shown to permeabilize the biomembranes in the tissue sections, it can be concluded that the rise in KM and Vmax values has to be attributed to the catalytic unit of the glucose-6-phosphatase system. It is suggested that the enzyme exists in a high affinity configuration at low enzyme concentrations but that at high enzyme concentrations a hysteretic mechanism, as proposed by Berteloot et al. (Berteloot, A., Vidal, H., and Van de Werve, G. (1991) J. Biol. Chem. 266, 5497-5507), transforms the enzyme from a high to a low affinity configuration. The present study indicates that the concept of functional heterogeneity of liver parenchyma may be more complex than thus far assumed.     

Tea catechol oxidase: Isolation,purification and kinetic characterization

Cathechol oxidase extracted from tea leaves was purified over 200-fold, using isoelectric focusing. The purified catechol oxidase was free of peroxidase and flavanol gallate esterase activities. Further, this enzyme was shown to have optimum activity near pH 5·7 and a K_m of 2·3 × 10^?3 M (at 25°) for (?)-epigallocatechin gallate. The purified enzyme was found to be capable of epimerizing tea flavanols at their C-2 position whether oxidation of the flavanol occurs (aerobic conditions) or not (anaerobic conditions). When oxygen is present, gallic acid is formed as a result of oxidation of either (?)-epigallocatechin gallate or (?)-epicatechin gallate. Formation of gallic acid is a side reaction of the oxidation of the flavanol gallates and is named oxidative degallation; no esterase per se is involved in this reaction.     

In situ measurements of viral particles diffusion inside mucoid biofilms

Fluorescence correlation spectroscopy (FCS) under two-photon excitation was used successfully to characterize the diffusion properties of model virus particles (bacteriophages) in bacterial biofilm of Stenotrophonas maltophilia. The results are compared to those obtained with fluorescent latex beads used as a reference. The FCS data clearly demonstrated the possibility for viral particles to penetrate inside the exopolymeric matrix of mucoid biofilms, and hence to benefit from its protective effect toward antimicrobials (antibiotics and biocides). Microbial biofilms should hence be considered as potential reservoirs of pathogenic viruses, and are probably responsible for numerous persistent viral infections.     

In situ microscopy for on-line determination of biomass

A sensor is presented, which allows on-line microscopic observation of microorganisms during fermentations in bioreactors. This sensor, an In Situ Microscope (ISM) consists of a direct-light microscope with a measuring chamber, integrated in a 25 mm stainless steel tube, two CCD-cameras, and two frame-grabbers. The data obtained are processed by an automatic image analysis system. The ISM is connected with the bioreactor via a standard port, and it is immersed directly in the culture liquid-in our case Saccharomyces cerevisiae in a synthetic medium. The microscopic examination of the liquid is performed in the measuring chamber, which is situated near the front end of the sensor head. The measuring chamber is opened and closed periodically. In the open state, the liquid in the bioreactor flows unrestricted through the chamber. In closing, a defined volume of 2,2. 10(-8) mL of the liquid becomes enclosed. After a few seconds, when the movement of the cells in the enclosed culture has stopped, they are examined with the microscope. The microscopic images of the cells are registered with the CCD-cameras and are visualized on a monitor, allowing a direct view of the cell population. After detection, the measuring chamber reopens, and the enclosed liquid is released. The images obtained are evaluated as to cell concentration, cell size, cell volume, biomass, and other relevant parameters simultaneously by automatic image analysis. With a PC (486/33 MHz), image processing takes about 15 s per image. The detection range tested when measuring cells of S. cerevisiae is about 10(6) to 10(9) cells/mL (equivalent to a biomass of 0.01 g/L to 12 g/L). The calculated biomass values correlate very well with those obtained using dry weight analysis. Furthermore, histograms can be calculated, which are comparable to those obtained by flow cytometry.     

In situ proteolysis for protein crystallization and structure determination

We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.     

In situ detection of bacteria in calcified biofilms using FISH and CARD-FISH

Modified protocols of fluorescence in situ hybridization (FISH) and catalyze reporter deposition fluorescence in situ hybridization (CARD-FISH) were developed in order to detect bacteria in situ in calcified stromatolite biofilms. Smooth, well-preserved thin sections of calcified biofilms (~5 microm thin, vertical sectioning of ~1 cm deep) were obtained by cryo-sectioning using the adhesive tape-stabilization technique. A modified hybridization buffer was applied during hybridization to prevent calcite dissolution as well as false binding of oligonucleotide probes to the charged mineral surfaces. Particularly, bright and specific CARD-FISH signals allowed the detection of bacteria in intensively calcified biofilms even at low magnification, which is suitable for investigating millimeter- to centimeter-scale vertical distribution patterns of bacteria.     

Methods for in situ detection and characterization of extracellular polymers in biofilms by electron microscopy

Electron microscopy of biofilms and the localization of extracellular polymers at high resolution require the adaptation of conventional electron microscopic preparation and imaging techniques. A method developed for in situ fixation and embedding of biofilms, imaging of unstained thick sections with electron spectroscopic imaging and the application of lectin or antibody-based marker systems allowed interpretation of extracellular polymer distribution at micrometer scale. By this way, it is possible to discriminate in situ between extracellular polymers produced by different organisms.     

Dynamics and control of biofilms of the oligotrophic bacterium Caulobacter crescentus

Caulobacter crescentus is an oligotrophic alpha-proteobacterium with a complex cell cycle involving sessile-stalked and piliated, flagellated swarmer cells. Because the natural lifestyle of C. crescentus intrinsically involves a surface-associated, sessile state, we investigated the dynamics and control of C. crescentus biofilms developing on glass surfaces in a hydrodynamic system. In contrast to biofilms of the well-studied Pseudomonas aeruginosa, Escherichia coli, and Vibrio cholerae, C. crescentus CB15 cells form biphasic biofilms, consisting predominantly of a cell monolayer biofilm and a biofilm containing densely packed, mushroom-shaped structures. Based on comparisons between the C. crescentus strain CB15 wild type and its holdfast (hfsA; DeltaCC0095), pili (DeltapilA-cpaF::Omegaaac3), motility (motA), flagellum (flgH) mutants, and a double mutant lacking holdfast and flagellum (hfsA; flgH), a model for biofilm formation in C. crescentus is proposed. For both biofilm forms, the holdfast structure at the tip of a stalked cell is crucial for mediating the initial attachment. Swimming motility by means of the single polar flagellum enhances initial attachment and enables progeny swarmer cells to escape from the monolayer biofilm. The flagellum structure also contributes to maintaining the mushroom structure. Type IV pili enhance but are not absolutely required for the initial adhesion phase. However, pili are essential for forming and maintaining the well-defined three-dimensional mushroom-shaped biofilm. The involvement of pili in mushroom architecture is a novel function for type IV pili in C. crescentus. These unique biofilm features demonstrate a spatial diversification of the C. crescentus population into a sessile, "stem cell"-like subpopulation (monolayer biofilm), which generates progeny cells capable of exploring the aqueous, oligotrophic environment by swimming motility and a subpopulation accumulating in large mushroom structures.     

Isolation, characterization, and in situ detection of a novel chemolithoautotrophic sulfur-oxidizing bacterium in wastewater biofilms growing under microaerophilic conditions

We successfully isolated a novel aerobic chemolithotrophic sulfur-oxidizing bacterium, designated strain SO07, from wastewater biofilms growing under microaerophilic conditions. For isolation, the use of elemental sulfur (S(0)), which is the most abundant sulfur pool in the wastewater biofilms, as the electron donor was an effective measure to establish an enrichment culture of strain SO07 and further isolation. 16S rRNA gene sequence analysis revealed that newly isolated strain SO07 was affiliated with members of the genus Halothiobacillus, but it was only distantly related to previously isolated species (89% identity). Strain SO07 oxidized elemental sulfur, thiosulfate, and sulfide to sulfate under oxic conditions. Strain SO07 could not grow on nitrate. Organic carbons, including acetate, propionate, and formate, could not serve as carbon and energy sources. Unlike other aerobic sulfur-oxidizing bacteria, this bacterium was sensitive to NaCl; growth in medium containing more than 150 mM was negligible. In situ hybridization combined with confocal laser scanning microscopy revealed that a number of rod-shaped cells hybridized with a probe specific for strain SO07 were mainly present in the oxic biofilm strata (ca. 0 to 100 micro m) and that they often coexisted with sulfate-reducing bacteria in this zone. These results demonstrated that strain SO07 was one of the important sulfur-oxidizing populations involved in the sulfur cycle occurring in the wastewater biofilm and was primarily responsible for the oxidation of H(2)S and S(0) to SO(4)(2-) under oxic conditions.     

A new multi-wavelength model-based method for determination of enzyme kinetic parameters

Lineweaver-Burk plot analysis is the most widely used method to determine enzyme kinetic parameters. In the spectrophotometric determination of enzyme activity using the Lineweaver-Burk plot, it is necessary to find a wavelength at which only the substrate or the product has absorbance without any spectroscopic interference of the other reaction components. Moreover, in this method, different initial concentrations of the substrate should be used to obtain the initial velocities required for Lineweaver-Burk plot analysis. In the present work, a multi-wavelength model-based method has been developed and validated to determine Michaelis-Menten constants for some enzyme reactions. In this method, a selective wavelength region and several experiments with different initial concentrations of the substrate are not required. The absorbance data of the kinetic assays are fitted by non-linear regression coupled to the numeric integration of the related differential equation. To indicate the applicability of the proposed method, the Michaelis-Menten constants for the oxidation of phenanthridine, 6-deoxypenciclovir and xanthine by molybdenum hydroxylases were determined using only a single initial concentration of the substrate, regardless of any spectral overlap.     

In vitro characterization of Trichophyton rubrum and T. mentagrophytes biofilms

Dermatophytes are fungi responsible for a disease known as dermatophytosis. Biofilms are sessile microbial communities surrounded by extracellular polymeric substances (EPS) with increased resistance to antimicrobial agents and host defenses. This paper describes, for the first time, the characteristics of Trichophyton rubrum and T. mentagrophytes biofilms. Biofilm formation was analyzed by light microscopy, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) as well as by staining with crystal violet and safranin. Metabolic activity was determined using the XTT reduction assay. Both species were able to form mature biofilms in 72?h. T. rubrum biofilm produced more biomass and EPS and was denser than T. mentagrophytes biofilm. The SEM results demonstrated a coordinated network of hyphae in all directions, embedded within EPS in some areas. Research and characterization of biofilms formed by dermatophytes may contribute to the search of new drugs for the treatment of these mycoses and might inform future revisions with respect to the dose and duration of treatment of currently available antifungals.     

Principle and method of kinetic microphotometric enzyme activity determination in situ

Summary An advanced apparative set-up is described for multipositional microphotometric recording of histochemical enzyme reactions in cryostat sections. It consists of a computer controlled microscope photometer with scanning stage. Measurements on the same tissue section may be performed at 12 preselected positions. These are repeatedly brought into the measuring beam in several measuring cycles. The complete measuring process, storage of measuring position coordinates, movements of the stage and statistical evaluation of the data is under computer control. By use of the gel film technique, extinction changes in tetrazolium coupled enzyme reactions can be measured continuously at initial rate conditions. Measurements are performed at identical conditions and can thus be analysed as relative enzyme activities.     

Principle and method of kinetic microphotometric enzyme activity determination in situ

An advanced apparative set-up is described for multipositional microphotometric recording of histochemical enzyme reactions in cryostat sections. It consists of a computer controlled microscope photometer with scanning stage. Measurements on the same tissue section may be performed at 12 preselected positions. These are repeatedly brought into the measuring beam in several measuring cycles. The complete measuring process, storage of measuring position coordinates, movements of the stage and statistical evaluation of the data is under computer control. By use of the gel film technique, extinction changes in tetrazolium coupled enzyme reactions can be measured continuously at initial rate conditions. Measurements are performed at identical conditions and can thus be analysed as relative enzyme activities.     

Laccase catalysed oxidation of syringic acid: calorimetric determination of kinetic parameters

Isothermal titration calorimetry (ITC) was used to study the oxidation of syringic acid by laccases from two different sources: Galerina sp. HC1 and Trametes versicolor. Total molar heat of reaction with both enzymes was similar (230 kJ/mol for Galerina laccase and 233 kJ/mol for Trametes laccase), and was independent of syringic acid concentration. The kinetic parameters of the reaction were calculated from the single injection assay by applying the nonlinear least squares fitting (NLSF) of experimental data to the Michaelis-Menten equation. Higher values for V(max) were obtained with Galerina sp. laccase, whereas K(m) values were comparable for the two enzymes.     

In situ analysis of nitrifying biofilms as determined by in situ hybridization and the use of microelectrodes.

We investigated the in situ spatial organization of ammonia-oxidizing and nitrite-oxidizing bacteria in domestic wastewater biofilms and autotrophic nitrifying biofilms by using microsensors and fluorescent in situ hybridization (FISH) performed with 16S rRNA-targeted oligonucleotide probes. The combination of these techniques made it possible to relate in situ microbial activity directly to the occurrence of nitrifying bacterial populations. In situ hybridization revealed that bacteria belonging to the genus Nitrosomonas were the numerically dominant ammonia-oxidizing bacteria in both types of biofilms. Bacteria belonging to the genus Nitrobacter were not detected; instead, Nitrospira-like bacteria were the main nitrite-oxidizing bacteria in both types of biofilms. Nitrospira-like cells formed irregularly shaped aggregates consisting of small microcolonies, which clustered around the clusters of ammonia oxidizers. Whereas most of the ammonia-oxidizing bacteria were present throughout the biofilms, the nitrite-oxidizing bacteria were restricted to the active nitrite-oxidizing zones, which were in the inner parts of the biofilms. Microelectrode measurements showed that the active ammonia-oxidizing zone was located in the outer part of a biofilm, whereas the active nitrite-oxidizing zone was located just below the ammonia-oxidizing zone and overlapped the location of nitrite-oxidizing bacteria, as determined by FISH.