Impact of biocontrol strain Pseudomonas fluorescens CHA0 on rhizosphere bacteria isolated from barley (Hordeum vulgare L.) with special reference to Cytophaga-like bacteria.

AIMS: To assess the impact of the biocontrol strain Pseudomonas fluorescens CHA0 on a collection of barley rhizosphere bacteria using an agar plate inhibition assay and a plant microcosm, focusing on a CHA0-sensitive member of the Cytophaga-like bacteria (CLB). METHODS AND RESULTS: The effect of strain CHA0 on a collection of barley rhizosphere bacteria, in particular CLB and fluorescent pseudomonads sampled during a growth season, was assessed by a growth inhibition assay. On average, 85% of the bacteria were sensitive in the May sample, while the effect was reduced to around 68% in the July and August samples. In the May sample, around 95% of the CLB and around 45% of the fluorescent pseudomonads were sensitive to strain CHA0. The proportion of CHA0-sensitive CLB and fluorescent pseudomonad isolates decreased during the plant growth season, i.e. in the July and August samples. A particularly sensitive CLB isolate, CLB23, was selected, exposed to strain CHA0 (wild type) and its genetically modified derivatives in the rhizosphere of barley grown in gnotobiotic soil microcosms. Two dry-stress periods were imposed during the experiment. Derivatives of strain CHA0 included antibiotic or exopolysaccharide (EPS) overproducing strains and a dry-stress-sensitive mutant. Despite their inhibitory activity against CLB23 in vitro, neither wild-type strain CHA0, nor any of its derivatives, had a major effect on culturable and total cell numbers of CLB23 during the 23-day microcosm experiment. Populations of all inoculants declined during the two dry-stress periods, with soil water contents below 5% and plants reaching the wilting point, but they recovered after re-wetting the soil. Survival of the dry-stress-sensitive mutant of CHA0 was most affected by the dry periods; however, this did not result in an increased population density of CLB23. CONCLUSIONS: CLB comprise a large fraction of barley rhizosphere bacteria that are sensitive to the biocontrol pseudomonad CHA0 in vitro. However, in plant microcosm experiments with varying soil humidity conditions, CHA0 or its derivatives had no major impact on the survival of the highly sensitive CLB strain, CLB23, during two dry-stress periods and a re-wetting period; all co-existed well in the rhizosphere of barley plants. SIGNIFICANCE AND IMPACT OF THE STUDY: Results indicate a lack of interaction between the biocontrol pseudomonad CHA0 and a sensitive CLB when the complexity increases from agar plate assays to plant microcosm experiments. This suggests the occurrence of low levels of antibiotic production and/or that the two bacterial genera occupy different niches in the rhizosphere.

Identification of active oxalotrophic bacteria by Bromodeoxyuridine DNA labeling in a microcosm soil experiments

The oxalate-carbonate pathway (OCP) leads to a potential carbon sink in terrestrial environments. This process is linked to the activity of oxalotrophic bacteria. Although isolation and molecular characterizations are used to study oxalotrophic bacteria, these approaches do not give information on the active oxalotrophs present in soil undergoing the OCP. The aim of this study was to assess the diversity of active oxalotrophic bacteria in soil microcosms using the Bromodeoxyuridine (BrdU) DNA labeling technique. Soil was collected near an oxalogenic tree (Milicia excelsa). Different concentrations of calcium oxalate (0.5%, 1%, and 4% w/w) were added to the soil microcosms and compared with an untreated control. After 12days of incubation, a maximal pH of 7.7 was measured for microcosms with oxalate (initial pH 6.4). At this time point, a DGGE profile of the frc gene was performed from BrdU-labeled soil DNA and unlabeled soil DNA. Actinobacteria (Streptomyces- and Kribbella-like sequences), Gammaproteobacteria and Betaproteobacteria were found as the main active oxalotrophic bacterial groups. This study highlights the relevance of Actinobacteria as members of the active bacterial community and the identification of novel uncultured oxalotrophic groups (i.e. Kribbella) active in soils.

Regulatory roles of the GacS/GacA two-component system in plant-associated and other gram-negative bacteria.

The sensor kinase GacS and the response regulator GacA are members of a two-component system that is present in a wide variety of gram-negative bacteria and has been studied mainly in enteric bacteria and fluorescent pseudomonads. The GacS/GacA system controls the production of secondary metabolites and extracellular enzymes involved in pathogenicity to plants and animals, biocontrol of soilborne plant diseases, ecological fitness, or tolerance to stress. A current model proposes that GacS senses a still-unknown signal and activates, via a phosphorelay mechanism, the GacA transcription regulator, which in turn triggers the expression of target genes. The GacS protein belongs to the unorthodox sensor kinases, characterized by an autophosphorylation, a receiver, and an output domain. The periplasmic loop domain of GacS is poorly conserved in diverse bacteria. Thus, a common signal interacting with this domain would be unexpected. Based on a comparison with the transcriptional regulator NarL, a secondary structure can be predicted for the GacA sensor kinases. Certain genes whose expression is regulated by the GacS/GacA system are regulated in parallel by the small RNA binding protein RsmA (CsrA) at a posttranscriptional level. It is suggested that the GacS/GacA system operates a switch between primary and secondary metabolism, with a major involvement of posttranscriptional control mechanisms.

Cell differentiation to "mating bodies" induced by an integrating and conjugative element in free-living bacteria.

Lateral gene transfer (LGT) is one of the most important processes leading to prokaryotic genome innovation. LGT is typically associated with conjugative plasmids and bacteriophages, but recently, a new class of mobile DNA known as integrating and conjugative elements (ICE) was discovered, which is abundant and widespread among bacterial genomes. By studying at the single-cell level the behavior of a prevalent ICE type in the genus Pseudomonas, we uncover the remarkable way in which the ICE orchestrates host cell differentiation to ensure horizontal transmission. We find that the ICE induces a state of transfer competence (tc) in 3%-5% of cells in a population under nongrowing conditions. ICE factors control the development of tc cells into specific assemblies that we name "mating bodies." Interestingly, cells in mating bodies undergo fewer and slower division than non-tc cells and eventually lyse. Mutations in ICE genes disrupting mating-body formation lead to 5-fold decreased ICE transfer rates. Hence, by confining the tc state to a small proportion of the population, ICE horizontal transmission is achieved with little cost in terms of vertical transmission. Given the low transfer frequencies of most ICE, we anticipate regulation by subpopulation differentiation to be widespread.

Human alveolar macrophages infected by virulent bacteria expressing SipB are a major source of active interleukin-18.

Recent publications have demonstrated that the protease caspase-1 is responsible for the processing of pro-interleukin 18 (IL-18) into the active form. Studies on cell lines and murine macrophages have shown that the bacterial invasion factor SipB activates caspase-1, triggering cell death. Thus, we investigated the role of SipB in the activation and release of IL-18 in human alveolar macrophages (AM), which are the first line of defense against inhaled pathogens. Under steady-state conditions, AM are a more important source of IL-18 than are dendritic cells (DC) and monocytes. Cytokine production by AM and DC was compared after both types of cells had been infected with a virulent strain of Salmonella enterica serovar Typhimurium and an isogenic sipB mutant, which were used as an infection model. Infection with virulent Salmonella led to marked cell death with features of apoptosis while both intracellular activation and release of IL-18 were demonstrated. In contrast, the sipB mutant did not induce such cell death or the release of active IL-18. The specific caspase-1 inhibitor Ac-YVAD-CMK blocked the early IL-18 release in AM infected with the virulent strain. However, the type of Salmonella infection did not differentially regulate IL-18 gene expression. We concluded that the bacterial virulence factor SipB plays an essential posttranslational role in the intracellular activation of IL-18 and the release of the cytokine in human AM.

Innate sensors for Gram-positive bacteria.

More than half of invasive bacterial infections are Gram-positive in origin. This class of bacteria has neither endotoxins nor an outer membrane, yet it generates some of the most powerful inflammatory responses known in medicine. Some recent seminal studies go a long way toward settling the controversies that surround the process by which Gram-positive bacterial surfaces trigger the human immune system. Although the components of the cell wall are now chemically defined in exquisite detail and the interaction with the toll-like receptor 2 pathway has been discovered, it is only very recently that definitive studies combining these advanced biochemical and cell biological tools have been carried out. It is these breakthrough studies that have finally confirmed the paradigm of innate sensors for Gram-positive bacteria.

Monosomal karyotype in acute myeloid leukemia: a better indicator of poor prognosis than a complex karyotype.

PURPOSE: To investigate the prognostic value of various cytogenetic components of a complex karyotype in acute myeloid leukemia (AML). PATIENTS AND METHODS: Cytogenetics and overall survival (OS) were analyzed in 1,975 AML patients age 15 to 60 years. RESULTS: Besides AML with normal cytogenetics (CN) and core binding factor (CBF) abnormalities, we distinguished 733 patients with cytogenetic abnormalities. Among the latter subgroup, loss of a single chromosome (n = 109) conferred negative prognostic impact (4-year OS, 12%; poor outcome). Loss of chromosome 7 was most common, but outcome of AML patients with single monosomy -7 (n = 63; 4-year OS, 13%) and other single autosomal monosomies (n = 46; 4-year OS, 12%) did not differ. Structural chromosomal abnormalities influenced prognosis only in association with a single autosomal monosomy (4-year OS, 4% for very poor v 24% for poor). We derived a monosomal karyotype (MK) as a predictor for very poor prognosis of AML that refers to two or more distinct autosomal chromosome monosomies (n = 116; 4-year OS, 3%) or one single autosomal monosomy in the presence of structural abnormalities (n = 68; 4-year OS, 4%). In direct comparisons, MK provides significantly better prognostic prediction than the traditionally defined complex karyotype, which considers any three or more or five or more clonal cytogenetic abnormalities, and also than various individual specific cytogenetic abnormalities (eg, del[5q], inv[3]/t[3;3]) associated with very poor outcome. CONCLUSION: MK enables (in addition to CN and CBF) the prognostic classification of two new aggregates of cytogenetically abnormal AML, the unfavorable risk MK-negative category (4-year OS, 26% +/- 2%) and the highly unfavorable risk MK-positive category (4-year OS, 4% +/- 1%).

Frequency-dependent attenuation as a potential indicator of oil saturation

At seismic frequencies, wave-induced fluid flow is a major cause of P-wave attenuation in partially saturated porous rocks. Attenuation is of great importance for the oil industry in the interpretation of seismic field data. Here, the effects on P-wave attenuation resulting from changes in oil saturation are studied for media with coexisting water, oil, and gas. For that, creep experiments are numerically simulated by solving Biot's equations for consolidation of poroelastic media with the finite-element method. The experiments yield time-dependent stress?strain relations that are used to calculate the complex P-wave modulus from which frequency-dependent P-wave attenuation is determined. The models are layered media with periodically alternating triplets of layers. Models consisting of triplets of layers having randomly varying layer thicknesses are also considered. The layers in each triplet are fully saturated with water, oil, and gas. The layer saturated with water has lower porosity and permeability than the layers saturated with oil and gas. These models represent hydrocarbon reservoirs in which water is the wetting fluid preferentially saturating regions of lower porosity. The results from the numerical experiments showed that increasing oil saturation, connected to a decrease in gas saturation, resulted in a significant increase of attenuation at low frequencies (lower than 2 Hz). Furthermore, replacing the oil with water resulted in a distinguishable behavior of the frequency-dependent attenuation. These results imply that, according to the physical mechanism of wave-induced fluid flow, frequency-dependent attenuation in media saturated with water, oil, and gas is a potential indicator of oil saturation.

Optimization of preservation conditions of As (III) bioreporter bacteria.

Long-term preservation of bioreporter bacteria is essential for the functioning of cell-based detection devices, particularly when field application, e.g., in developing countries, is intended. We varied the culture conditions (i.e., the NaCl content of the medium), storage protection media, and preservation methods (vacuum drying vs. encapsulation gels remaining hydrated) in order to achieve optimal preservation of the activity of As (III) bioreporter bacteria during up to 12 weeks of storage at 4 degrees C. The presence of 2% sodium chloride during the cultivation improved the response intensity of some bioreporters upon reconstitution, particularly of those that had been dried and stored in the presence of sucrose or trehalose and 10% gelatin. The most satisfying, stable response to arsenite after 12 weeks storage was obtained with cells that had been dried in the presence of 34% trehalose and 1.5% polyvinylpyrrolidone. Amendments of peptone, meat extract, sodium ascorbate, and sodium glutamate preserved the bioreporter activity only for the first 2 weeks, but not during long-term storage. Only short-term stability was also achieved when bioreporter bacteria were encapsulated in gels remaining hydrated during storage.

Conviction Statistics as an Indicator of Crime Trends in Europe from 1990 to 2006

Convictions statistics were the first criminal statistics available in Europe during the nineteenth century. Their main weaknesses as crime measures and for comparative purposes were identified by Alphonse de Candolle in the 1830s. Currently, they are seldom used by comparative criminologists, although they provide a less valid but more reliable measure of crime and formal social control than police statistics. This article uses conviction statistics, compiled from the four editions of the European Sourcebook of Crime and Criminal Justice Statistics, to study the evolution of persons convicted in European countries from 1990 to 2006. Trends in persons convicted for six offences -intentional homicide, assault, rape, robbery, theft, and drug offences- and up to 26 European countries are analysed. These trends are established for the whole of Europe as well as for a cluster of Western European countries and a cluster of Central and Eastern European countries. The analyses show similarities between both regions of Europe at the beginning and at the end of the period under study. After a general increase of the rate of persons convicted in the early 1990s in the whole of Europe, trends followed different directions in Western and in Central and Eastern Europe. However, during the 2000s, it can be observed, throughout Europe, a certain stability of the rates of persons convicted for intentional homicides, accompanied by a general decrease of the rate of persons convicted for property offences, and an increase of the rate of those convicted for drug offences. The latter goes together with an increase of the rate of persons convicted for non lethal violent offences, which only reached some stability at the end of the time series. These trends show that there is no general crime drop in Europe. After a discussion of possible theoretical explanations, a multifactor model, inspired by opportunity-based theories, is proposed to explain the trends observed.

Small, stable shuttle vectors based on the minimal pVS1 replicon for use in gram-negative, plant-associated bacteria.

The minimal replicon of the Pseudomonas plasmid pVS1 was genetically defined and combined with the Escherichia coli p15A replicon, to provide a series of new, oligocopy cloning vectors (5.3 to 8.3 kb). Recombinant plasmids derived from these vectors were stable in growing and nongrowing cells of root-colonizing P. fluorescens strains incubated under different environmental conditions for more than 1 month.

Detection of live and antibiotic-killed bacteria by quantitative real-time PCR of specific fragments of rRNA.

Assessing bacterial viability by molecular markers might help accelerate the measurement of antibiotic-induced killing. This study investigated whether rRNA could be suitable for this purpose. Cultures of penicillin-susceptible and penicillin-tolerant (Tol1 mutant) Streptococcus gordonii were exposed to mechanistically different penicillin and levofloxacin. Bacterial survival was assessed by viable counts and compared to quantitative real-time PCR amplification of either the 16S rRNA genes or the 16S rRNA, following reverse transcription. Penicillin-susceptible S. gordonii lost > or =4 log(10) CFU/ml of viability over 48 h of penicillin treatment. In comparison, the Tol1 mutant lost < or =1 log(10) CFU/ml. Amplification of a 427-bp fragment of 16S rRNA genes yielded amplicons that increased proportionally to viable counts during bacterial growth but did not decrease during drug-induced killing. In contrast, the same 427-bp fragment amplified from 16S rRNA paralleled both bacterial growth and drug-induced killing. It also differentiated between penicillin-induced killing of the parent and the Tol1 mutant (> or =4 log(10) CFU/ml and < or =1 log(10) CFU/ml, respectively) and detected killing by mechanistically unrelated levofloxacin. Since large fragments of polynucleotides might be degraded faster than smaller fragments, the experiments were repeated by amplifying a 119-bp region internal to the original 427-bp fragment. The amount of 119-bp amplicons increased proportionally to viability during growth but remained stable during drug treatment. Thus, 16S rRNA was a marker of antibiotic-induced killing, but the size of the amplified fragment was critical for differentiation between live and dead bacteria.

Plants respond to pathogen infection by enhancing the antifungal gene expression of root-associated bacteria.

Plant health and fitness widely depend on interactions with soil microorganisms. Some bacteria such as pseudomonads can inhibit pathogens by producing antibiotics, and controlling these bacteria could help improve plant fitness. In the present study, we tested whether plants induce changes in the antifungal activity of root-associated bacteria as a response to root pathogens. We grew barley plants in a split-root system with one side of the root system challenged by the pathogen Pythium ultimum and the other side inoculated with the biocontrol strain Pseudomonas fluorescens CHA0. We used reporter genes to follow the expression of ribosomal RNA indicative of the metabolic state and of the gene phlA, required for production of 2,4-diacetylphloroglucinol, a key component of antifungal activity. Infection increased the expression of the antifungal gene phlA. No contact with the pathogen was required, indicating that barley influenced gene expression by the bacteria in a systemic way. This effect relied on increased exudation of diffusible molecules increasing phlA expression, suggesting that communication with rhizosphere bacteria is part of the pathogen response of plants. Tripartite interactions among plants, pathogens, and bacteria appear as a novel determinant of plant response to root pathogens.