Impact of protozoan grazing on bacterial community structure in soil microcosms

The influence of grazing by a mixed assemblage of soil protozoa (seven flagellates and one amoeba) on bacterial community structure was studied in soil microcosms amended with a particulate resource (sterile wheat roots) or a soluble resource (a solution of various organic compounds). Sterilized soil was reinoculated with mixed soil bacteria (obtained by filtering and dilution) or with bacteria and protozoa. Denaturing gradient gel electrophoresis (DGGE) of PCR amplifications of 16S rRNA gene fragments, as well as community level physiological profiling (Biolog plates), suggested that the mixed protozoan community had significant effects on the bacterial community structure. Excising and sequencing of bands from the DGGE gels indicated that high-G+C gram-positive bacteria closely related to Arthrobacter spp. were favored by grazing, whereas the excised bands that decreased in intensity were related to gram-negative bacteria. The percentages of intensity found in bands related to high G+C gram positives increased from 4.5 and 12.6% in the ungrazed microcosms amended with roots and nutrient solution, respectively, to 19.3 and 32.9% in the grazed microcosms. Protozoa reduced the average bacterial cell size in microcosms amended with nutrient solution but not in the treatment amended with roots. Hence, size-selective feeding may explain some but not all of the changes in bacterial community structure. Five different protozoan isolates (Acanthamoeba sp., two species of Cercomonas, Thaumatomonas sp., and Spumella sp.) had different effects on the bacterial communities. This suggests that the composition of protozoan communities is important for the effect of protozoan grazing on bacterial communities.     

The role of quorum sensing mediated developmental traits in the resistance of Serratia marcescens biofilms against protozoan grazing

Resistance against protozoan grazers is a crucial factor that is important for the survival of many bacteria in their natural environment. However, the basis of resistance to protozoans and how resistance factors are regulated is poorly understood. In part, resistance may be due to biofilm formation, which is known to protect bacteria from environmental stress conditions. The ubiquitous organism Serratia marcescens uses quorum sensing (QS) control to regulate virulence factor expression and biofilm formation. We hypothesized that the QS system of S. marcescens also regulates mechanisms that protect biofilms against protozoan grazing. To investigate this hypothesis, we compared the interactions of wild-type and QS mutant strains of S. marcescens biofilms with two protozoans having different feeding types under batch and flow conditions. Under batch conditions, S. marcescens forms microcolony biofilms, and filamentous biofilms are formed under flow conditions. The microcolony-type biofilms were protected from grazing by the suspension feeder, flagellate Bodo saltans, but were not protected from the surface feeder, Acanthamoeba polyphaga. In contrast, the filamentous biofilm provided protection against A. polyphaga. The main findings presented in this study suggest that (i) the QS system is not involved in grazing resistance of S. marcescens microcolony-type biofilms; (ii) QS in S. marcescens regulates antiprotozoan factor(s) that do not interfere with the grazing efficiency of the protozoans; and (iii) QS-controlled, biofilm-specific differentiation of filaments and cell chains in biofilms of S. marcescens provides an efficient mechanism against protozoan grazing.     

Morphological and compositional changes in a planktonic bacterial community in response to enhanced protozoan grazing

We analyzed changes in bacterioplankton morphology and composition during enhanced protozoan grazing by image analysis and fluorescent in situ hybridization with group-specific rRNA-targeted oligonucleotide probes. Enclosure experiments were conducted in a small, fishless freshwater pond which was dominated by the cladoceran Daphnia magna. The removal of metazooplankton enhanced protozoan grazing pressure and triggered a microbial succession from fast-growing small bacteria to larger grazing-resistant morphotypes. These were mainly different types of filamentous bacteria which correlated in biomass with the population development of heterotrophic nanoflagellates (HNF). Small bacterial rods and cocci, which showed increased proportion after removal of Daphnia and doubling times of 6 to 11 h, belonged nearly exclusively to the beta subdivision of the class Proteobacteria and the Cytophaga-Flavobacterium cluster. The majority of this newly produced bacterial biomass was rapidly consumed by HNF. In contrast, the proportion of bacteria belonging to the gamma and alpha subdivisions of the Proteobacteria increased throughout the experiment. The alpha subdivision consisted mainly of rods that were 3 to 6 microm in length, which probably exceeded the size range of bacteria edible by protozoa. Initially, these organisms accounted for less than 1% of total bacteria, but after 72 h they became the predominant group of the bacterial assemblage. Other types of grazing-resistant, filamentous bacteria were also found within the beta subdivision of Proteobacteria and the Cytophaga-Flavobacterium cluster. We conclude that the predation regimen is a major structuring force for the bacterial community composition in this system. Protozoan grazing resulted in shifts of the morphological as well as the taxonomic composition of the bacterial assemblage. Grazing-resistant filamentous bacteria can develop within different phylogenetic groups of bacteria, and formerly underepresented taxa might become a dominant group when protozoan predation is the major selective pressure.     

A new method for evaluating defense of microalgae against protozoan grazing

Body-size spectrum has proved to be a highly informative indicator to summarize the functional structure of a community at taxon-free resolution. In this study, an approach based on body-size spectrum of protozoan communities was used to detect the defense of microalgae against protozoan grazing. The biofilm-dwelling protozoan communities were used as a test predator system, and two algal species, Chlorella sp. and Nannochloropsis oceanica, were employed as test microalgae. A nine-day bioassay test was carried out by exposing biofilm-dwelling protozoan communities to a gradient of concentrations 10⁰ (control), 10⁴, 10⁵, 10⁶, and 10⁷ cell ml⁻¹ of both microalgae, respectively. Results showed that both algal species represented strong defense effects on the test predator system at different levels of concentration. The body-size distinctness of the protozoan assemblages showed a sharp decrease at high concentration level more than 10⁶ cell ml⁻¹ in both algal treatments. Based on the paired body-size distinctness indices of the protozoa, ellipse tests demonstrated that the body-size spectrum showed an increasing trend of departure from the expected pattern with increasing concentrations of both test algae. Thus, it is suggested that the body-size spectrum of protozoa may be used as a useful indicator to identify the defense of microalgae against protozoan grazing.     

Contribution of virus-induced lysis and protozoan grazing to benthic bacterial mortality estimated simultaneously in microcosms

In contrast to the water column, the fate of bacterial production in freshwater sediments is still a matter of debate. Thus, the importance of virus-induced lysis and protozoan grazing of bacteria was investigated for the first time simultaneously in a silty sediment layer of a mesotrophic oxbow lake. Microcosms were installed in the laboratory in order to study the dynamics of these processes over 15 days. All microbial and physicochemical parameters showed acceptable resemblance to field data observed during a concomitant in situ study, and similar conclusions can be drawn with respect to the quantitative impact of viruses and protozoa on the bacterial compartment. Viral decay rates ranged from undetectable to 0.078 h⁻¹ (average, 0.033 h⁻¹), and the control of bacterial production from below the detection limit to 36% (average, 12%). The contribution of virus-induced lysis of bacteria to the dissolved organic matter pool as well as to benthic bacterial nutrition was low. Ingestion rates of protozoan grazers ranged from undetectable to 24.7 bacteria per heterotrophic nanoflagellate (HNF) per hour (average, 4.8 bacteria HNF⁻¹ h⁻¹) and from undetectable to 73.3 bacteria per ciliate per hour (average, 11.2 bacteria ciliate⁻¹ h⁻¹). Heterotrophic nanoflagellate and ciliates together cropped up to 5% (average, 1%) of bacterial production. The viral impact on bacteria prevailed over protozoan grazing by a factor of 2.5–19.9 (average, 9.5). In sum, these factors together removed up to 36% (average, 12%) of bacterial production. The high number of correlations between viral and protozoan parameters is discussed in view of a possible relationship between virus removal and the presence of protozoan grazers.     

Nitrification in activated sludge batch reactors is linked to protozoan grazing of the bacterial population

Protozoa feed upon free-swimming bacteria and suspended particles inducing flocculation and increasing the turnover rate of nutrients in complex mixed communities. In this study, the effect of protozoan grazing on nitrification was examined in activated sludge in batch cultures maintained over a 14-day period. A reduction in the protozoan grazing pressure was accomplished by using either a dilution series or the protozoan inhibitor cycloheximide. As the dilutions increased, the nitrification rate showed a decline, suggesting that a reduction in protozoan or bacterial concentration may cause a decrease in nitrification potential. In the presence of cycloheximide, where the bacterial concentration was not altered, the rates of production of ammonia, nitrite, and nitrate all were significantly lower in the absence of active protozoans. These results suggest that a reduction in the number or activity of the protozoans reduces nitrification, possibly by limiting the availability of nutrients for slow-growing ammonia and nitrite oxidizers through excretion products. Furthermore, the ability of protozoans to groom the heterotrophic bacterial population in such systems may also play a role in reducing interspecies competition for nitrification substrates and thereby augment nitrification rates.     

A novel thermotropic liquid crystalline – Benzoylated bacterial cellulose

Using the esterification of bacterial cellulose (BC), we have synthesized Benzoylated bacterial cellulose (BBC). The molecular structure of the BBC was characterized by means of Fourier transform infrared (FT-IR) spectroscopy, ¹H and ¹³C nuclear magnetic resonance (NMR). The BBC is found to display thermotropic liquid crystalline feature determined with differential scanning calorimetry (DSC), polarized optical microscope (POM) and wide-angle X-ray diffraction (WAXD). Here, we demonstrate that it is possible to obtain the BBC with degree of substitution (DS) from 0.88 to 2.46 by applying the different molar ratio of benzoyl chloride to the anhydrous glucose unit (AGU). The glass transition temperatures (T_g) of the liquid crystalline phases lie between 281.2 and 281.8 °C and the isotropic melt transition temperatures (T_i) vary from 341.6 to 362.8 °C, depending on the DS.     

NetPhosBac – A predictor for Ser/Thr phosphorylation sites in bacterial proteins

There is ample evidence for the involvement of protein phosphorylation on serine/threonine/tyrosine in bacterial signaling and regulation, but very few exact phosphorylation sites have been experimentally determined. Recently, gel‐free high accuracy MS studies reported over 150 phosphorylation sites in two bacterial model organisms Bacillus subtilis and Escherichia coli. Interestingly, the analysis of these phosphorylation sites revealed that most of them are not characteristic for eukaryotic‐type protein kinases, which explains the poor performance of eukaryotic data‐trained phosphorylation predictors on bacterial systems. We used these large bacterial datasets and neural network algorithms to create the first bacteria‐specific protein phosphorylation predictor: NetPhosBac. With respect to predicting bacterial phosphorylation sites, NetPhosBac significantly outperformed all benchmark predictors. Moreover, NetPhosBac predictions of phosphorylation sites in E. coli proteins were experimentally verified on protein and site‐specific levels. In conclusion, NetPhosBac clearly illustrates the advantage of taxa‐specific predictors and we hope it will provide a useful asset to the microbiological community.     

Impact of protozoan grazing on nitrification and the ammonia- and nitrite-oxidizing bacterial communities in activated sludge

In activated sludge, protozoa feed on free-swimming bacteria and suspended particles, inducing flocculation and increasing the turnover rate of nutrients. In this study, the effect of protozoan grazing on nitrification rates under various conditions in municipal activated sludge batch reactors was examined, as was the spatial distribution of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) within the activated sludge. The reactors were monitored for ammonia, nitrite, nitrate, and total nitrogen concentrations, and bacterial numbers in the presence and absence of cycloheximide (a protozoan inhibitor), allylthiourea (an inhibitor of ammonia oxidation), and EDTA (a deflocculating agent). The accumulations of nitrate, nitrite, and ammonia were lower in batches without than with protozoa grazing. Inhibition of ammonia oxidation also decreased the amount of nitrite and nitrate accumulation. Inhibiting protozoan grazing along with ammonia oxidation further decreased the amounts of nitrite and nitrate accumulated. Induction of deflocculation led to high nitrate accumulation, indicating high levels of nitrification; this effect was lessened in the absence of protozoan grazing. Using fluorescent in situ hybridization and confocal laser scanning microscopy, AOB and NOB were found clustered within the floc, and inhibiting the protozoa, inhibiting ammonia oxidation, or inducing flocculation did not appear to lower the number of AOB and NOB present or affect their position within the floc. These results suggest that the AOB and NOB are present but less active in the absence of protozoa.     

Competition of two suspension-feeding protozoan populations for a growing bacterial population in continuous culture

Mathematical studies for ecosystems involving 2 predators competing for a growing prey population have shown that the 2 competitors can coexist in a state of sustained oscillations for a range of values of the system parameters. For the case of 1 suspension-feeding protozoan population, recent experimental observations suggest that the predator-prey interaction is complicated by the ability of the bacteria to grow on products produced by the lysis of protozoan cells. This situation is studied here for the case where 2 suspension-feeding protozoan populations compete for a growing bacterial population in a chemostat. Computer simulations show that the 2 protozoan populations can coexist over a range of the operating parameters. Some necessary conditions for coexistence are presented as are some speculations regarding the possible physical explanations of results.     

Seasonal and successional influences on bacterial community composition exceed that of protozoan grazing in river biofilms

The effects of protozoa (heterotrophic flagellates and ciliates) on the morphology and community composition of bacterial biofilms were tested under natural background conditions by applying size fractionation in a river bypass system. Confocal laser scanning microscopy (CLSM) was used to monitor the morphological structure of the biofilm, and fingerprinting methods (single-stranded conformation polymorphism [SSCP] and denaturing gradient gel electrophoresis [DGGE]) were utilized to assess changes in bacterial community composition. Season and internal population dynamics had a greater influence on the bacterial biofilm than the presence of protozoa. Within this general framework, bacterial area coverage and microcolony abundance were nevertheless enhanced by the presence of ciliates (but not by the presence of flagellates). We also found that the richness of bacterial operational taxonomic units was much higher in planktonic founder communities than in the ones establishing the biofilm. Within the first 2 h of colonization of an empty substrate by bacteria, the presence of flagellates additionally altered their biofilm community composition. As the biofilms matured, the number of bacterial operational taxonomic units increased when flagellates were present in high abundances. The additional presence of ciliates tended to at first reduce (days 2 to 7) and later increase (days 14 to 29) bacterial operational taxonomic unit richness. Altogether, the response of the bacterial community to protozoan grazing pressure was small compared to that reported in planktonic studies, but our findings contradict the assumption of a general grazing resistance of bacterial biofilms toward protozoa.     

A role for β‐sitosterol to stigmasterol conversion in plant–pathogen interactions

Upon inoculation with pathogenic microbes, plants induce an array of metabolic changes that potentially contribute to induced resistance or even enhance susceptibility. When analysing leaf lipid composition during the Arabidopsis thaliana–Pseudomonas syringae interaction, we found that accumulation of the phytosterol stigmasterol is a significant plant metabolic process that occurs upon bacterial leaf infection. Stigmasterol is synthesized from β‐sitosterol by the cytochrome P450 CYP710A1 via C22 desaturation. Arabidopsis cyp710A1 mutant lines impaired in pathogen‐inducible expression of the C22 desaturase and concomitant stigmasterol accumulation are more resistant to both avirulent and virulent P. syringae strains than wild‐type plants, and exogenous application of stigmasterol attenuates this resistance phenotype. These data indicate that induced sterol desaturation in wild‐type plants favours pathogen multiplication and plant susceptibility. Stigmasterol formation is triggered through perception of pathogen‐associated molecular patterns such as flagellin and lipopolysaccharides, and through production of reactive oxygen species, but does not depend on the salicylic acid, jasmonic acid or ethylene defence pathways. Isolated microsomal and plasma membrane preparations exhibited a similar increase in the stigmasterol/β‐sitosterol ratio as whole‐leaf extracts after leaf inoculation with P. syringae, indicating that the stigmasterol produced is incorporated into plant membranes. The increased contents of stigmasterol in leaves after pathogen attack do not influence salicylic acid‐mediated defence signalling but attenuate pathogen‐induced expression of the defence regulator flavin‐dependent monooxygenase 1. P. syringae thus promotes plant disease susceptibility through stimulation of sterol C22 desaturation in leaves, which increases the stigmasterol to β‐sitosterol ratio in plant membranes.     

Detecting variability and selecting for pesticide resistance in two species of phytoseiid mites

Methods used for evaluating the effects of pesticides and selecting for pesticide resistance in phytoseiid mites are reviewed from recent literature. In particular slide dip, leaf dip, and leaf disc spray methods are compared. The selection of predatory mites (Typhlodromus pyri Scheuten andPhytoseiulus persimilis Athias-Henriot) for resistance to 3 synthetic pyrethroids (SP-cypermethrin, deltamethrin, and fenvalerate) is described. Tolerance of field populations to all 3 SP was low inP. persimilis but moderate inT. pyri. Field samples of both mite species on leaf discs were sprayed and the survivors were reared in laboratory and/or glasshouse cultures. These cultures were sprayed with repeated doses of SP; initiallyT. pyri was selected with cypermethrin andP. persimilis with fenvalerate. The survival rate ofT. pyri increased at each selection. After 6 selections the survival rate of the laboratory culture was 10 times that of the original field samples. Tests indicated crossresistance inT. pyri to fenvalerate and deltamethrin. Selection with cypermethrin is continuing. In the first 12 months repeated selections ofP. persimilis with fenvalerate gave no significant change in survival rate.

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Résumé Les méthodes utilisées pour évaluer les effets des pesticides et pour sélectionner la résistance à ces mêmes produits, des acariens phytoseiides sont analysées d'après la littérature récente. La sélection des acariens prédateurs (Typhlodromus pyri S{upcheuten} etPhytoseiulus persimilis A{upthias}-H{upenriot}) pour leur résistance aux 3 pyréthrinoides de synthèse (cyperméthrine, deltaméthrine et fenvalerate) est décrite. La tolérance des populations naturelles aux 3 pyréthrinoides de synthèse était basse pourP. persimilis, mais modérée pourT. pyri. Les échantillons des 2 espèces d'acariens prélevés à l'extérieur furent traitès sur des disques de feuilles et les survivants furent élevés au laboratoire et/ou dans des cultures en serre. Ces élevages furent traités avec des doses répétées d'un pyréthrinoide, cyperméthrine initialement pourT. pyri et fenvalerate pourP. persimilis. Le taux de survie deT. pyri augmentait à chaque sélection. Après 6 sélections, le taux de survie de l'élevage de laboratoire était 10 fois celui des échantillons d'origine. Les essais révélaient une résistance croisée deT. pyri à la fenvalerate et à la deltaméthrine. La sélection avec la cyperméthrine se poursuit. Au cours des 12 premiers mois, les sélections répétées deP. persimilis avec la fenvalerate ne donnait pas de changement significatif dans le taux de survie.

     

‘Nothing is permanent but change’† – antigenic variation in persistent bacterial pathogens

Pathogens persist in immunocompetent mammalian hosts using various strategies, including evasion of immune effectors by antigenic variation. Among highly antigenically variant bacteria, gene conversion is used to generate novel expressed variants from otherwise silent donor sequences. Recombination using oligonucleotide segments from multiple donors is a combinatorial mechanism that tremendously expands the variant repertoire, allowing thousands of variants to be generated from a relatively small donor pool. Three bacterial pathogens, each encoded by a small genome (< 1.2 Mb), illustrate this variant generating capacity and its role in persistent infection. Borrelia burgdorferi VlsE diversity is encoded and expressed on a linear plasmid required for persistence and recent experiments have demonstrated that VlsE recombination is necessary for persistence in the immunocompetent host. In contrast, both Treponema pallidum TprK and Anaplasma marginale Msp2 expression sites and donors are chromosomally encoded. Both T. pallidum and A. marginale generate antigenic variants in vivo in individual hosts and studies at the population level reveal marked strain diversity in the variant repertoire that may underlie pathogen strain structure and the capacity for re‐infection and heterologous strain superinfection. Here, we review gene conversion in bacterial antigenic variation and discuss the short‐ and long‐term selective pressures that shape the variant repertoire.     

The role of the CD39–CD73–adenosine pathway in liver disease

Extracellular adenosine triphosphate (ATP) is a danger signal released by dying and damaged cells, and it functions as an immunostimulatory signal that promotes inflammation. The ectonucleotidases CD39/ectonucleoside triphosphate diphosphohydrolase‐1 and CD73/ecto‐5′‐nucleotidase are cell‐surface enzymes that breakdown extracellular ATP into adenosine. This drives a shift from an ATP‐driven proinflammatory environment to an anti‐inflammatory milieu induced by adenosine. The CD39–CD73–adenosine pathway changes dynamically with the pathophysiological context in which it is embedded. Accumulating evidence suggests that CD39 and CD73 play important roles in liver disease as critical components of the extracellular adenosinergic pathway. Recent studies have shown that the modification of the CD39–CD73–adenosine pathway alters the liver's response to injury. Moreover, adenosine exerts different effects on the pathophysiology of the liver through different receptors. In this review, we aim to describe the role of the CD39–CD73–adenosine pathway and adenosine receptors in liver disease, highlighting potential therapeutic targets in this pathway, which will facilitate the development of therapeutic strategies for the treatment of liver disease.     

Central role for cAMP signaling in pigmentation and UV resistance

Comment on: Khaled M, et al. Genes Dev 2010; 24:2276-81.     

Microselection – affinity selecting antibodies against a single rare cell in a heterogeneous population

Rare cells not normally present in the peripheral bloodstream, such as circulating tumour cells, have potential applications for development of non‐invasive methods for diagnostics or follow up. Obtaining these cells however require some means of discrimination, achievable by cell type specific antibodies. Here we have generated a microselection method allowing antibody selection, by phage display, targeting a single cell in a heterogeneous population. One K562 cell (female origin) was positioned on glass slide among millions of lymphocytes from male donor, identifying the K562 cell by FISH (XX). Several single cell selections were performed on such individual slides. The phage particles bound to the target cell is protected by a minute disc, while inactivating all remaining phage by UV‐irradiation; leaving only the phage bound to the target cell viable. We hereby retrieved up to eight antibodies per single cell selection, including three highly K562 cell type specific.     

Adventures with Bacillus megaterium – fusion of bacterial protoplasts

This essay describes the author's studies with bacteria in post-war Hungary; the difficulties encountered, with funding, collaboration and publication; and how the Szeged Institute consolidated itseif as the one outstanding scientific Institute in Eastern Europe, with the author at the helm.     

A role for copper in biological time-keeping

A family of cell surface and growth related proteins that oxidize both NADH and hydroquinones and carry out protein disulfide-thiol interchange (ECTO-NOX proteins) exhibits unique characteristics. The two activities they catalyze, hydroquinone or NADH oxidation and protein disulfide-thiol interchange, alternate in CNOX (the constitutive ECTO-NOX), to generate a regular period length of 24 min. For NADH or hydroquinone oxidation each period is defined by maxima that recur at intervals of 24 min. Here, we report that bound Cu^II is required to sustain the 24 min oscillation cycle of CNOX. CNOX preparations from plasma membranes of soybean, when unfolded in the presence of the copper chelator bathocuproine and refolded, lose activity. When refolded in the presence of copper, activity is restored. Unexpectedly, however, the released copper is capable of catalyzing NADH (or hydroquinone) oxidation in the absence of protein. Solvated Cu^II as the chloride or other salts alone is capable of catalyzing NADH oxidation and the oxidation rates oscillate with an overall period length of 24 min. With Cu^IICl₂ the pattern consists of five maxima, two of which are separated by an interval of 6 min and three of which are separated by intervals of 4.5 min [6 min + 4 (4.5 min)]. The period length is independent of temperature and pH. The asymmetry of the oscillatory pattern is retained after solvation of the Cu^II salts in D₂O but the overall period length is increased to 30 min. The findings suggest that the bound copper of CNOX and perhaps of ECTO-NOX proteins in general, is essential to maintain the structural changes that underlie the periodic alternations in activity that define the 24 min time-keeping cycle of the protein.