Characterization of the new human pleomorphic undifferentiated sarcoma <Emphasis Type="Italic">TP53</Emphasis>-<Emphasis Type="Italic">null</Emphasis> cell line <Emphasis Type="Italic">mfh</Emphasis>-<Emphasis Type="Italic">val2</Emphasis>

Pleomorphic undifferentiated sarcoma (PUS), also called malignant fibrous histiocytoma, is a soft tissue sarcoma which occurs predominantly in the extremities. Its origin is a poorly defined mesenchymal cell, which derives to histiocytic and fibroblastic cells. The patient, a 58 year-old man, presented a lesion located in the forearm composed by spindle cells and multinucleated giant cells, which expressed vimentin and adopted a histological pattern formed by irregular-swirling fascicles. Cells were cultured in vitro and a new cell line was established. We characterized this new cell line by histological analyses, cytogenetics (using G-bands and spectral karyotype technique) and cytometric analyses. Cells were grown in culture for more than 100 passages. They had elongated or polygonal morphology. The cells presented a saturation rate of 70,980 cells/cm², a plating efficiency of 21.5% and a mitotic index of 21 mitoses per field. The cell line was tumorigenic in nude mice. The ploidy study using flow cytometry revealed an aneuploid peak with a DNA index of 1.43. A side population was detected, demonstrating the presence of stem and progenitor cells. Cytogenetics showed a hypotriploid range with many clonal unbalanced rearrangements. Loss of p53 gene was evidenced by MLPA. We describe, for the first time, the characterization of a new human PUS TP53-null cell line called mfh-val2. Mfh-val2 presents a wide number of applications as a TP53-null cell line and a great interest in order to characterize genetic alterations influencing the oncogenesis or progression of PUS and to advance in the biological investigation of this tumor.     

<Emphasis Type="Italic">Drosophila melanogaster</Emphasis> as a polymicrobial infection model for <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Non-mammalian infection models have been developed over the last two decades, which is a historic milestone to understand the molecular basis of bacterial pathogenesis. They also provide small-scale research platforms for identification of virulence factors, screening for antibacterial hits, and evaluation of antibacterial efficacy. The fruit fly, Drosophila melanogaster is one of the model hosts for a variety of bacterial pathogens, in that the innate immunity pathways and tissue physiology are highly similar to those in mammals. We here present a relatively simple protocol to assess the key aspects of the polymicrobial interaction in vivo between the human opportunistic pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, which is based on the systemic infection by needle pricking at the dorsal thorax of the flies. After infection, fly survival and bacteremia over time for both P. aeruginosa and S. aureus within the infected flies can be monitored as a measure of polymicrobial virulence potential. The infection takes ~24 h including bacterial cultivation. Fly survival and bacteremia are assessed using the infected flies that are monitored up to ~60 h post-infection. These methods can be used to identify presumable as well as unexpected phenotypes during polymicrobial interaction between P. aeruginosa and S. aureus mutants, regarding bacterial pathogenesis and host immunity.     

Rapid detection of aneuploidy in <Emphasis Type="Italic">Musa</Emphasis> using flow cytometry

We report a procedure for the rapid and convenient detection of aneuploidy in triploid Musa using DNA flow cytometry. From a population of plants derived from gamma-irradiated shoot tips, plants were selected based on aberrant morphology and their chromosome numbers were counted. Aneuploids plants with chromosome numbers 2n=31 or 32 were found as well as the expected triploid plants (2n=3x=33). At the same time, the nuclear DNA content of all plants was measured using flow cytometry. The flow cytometric assay involved the use of nuclei isolated from chicken red blood cells (CRBC), which served as an internal reference standard. The relative DNA content of individual plants was expressed as a ratio of DNA content of CRBC and Musa (DNA index). In order to estimate the chromosome number using flow cytometry, the relative DNA content of plants with unknown ploidy was expressed as a percentage of the DNA content of triploid plants. The classification based on flow cytometry fully agreed with the results obtained by chromosome counting. The results indicated that flow cytometry is a convenient and rapid method for the detection of aneuploidy in Musa.     

Flow Cytometry as a Rapid Test for Detection of Penicillin Resistance Directly in Bacterial Cells in <Emphasis Type="Italic">Enterococcus faecalis</Emphasis> and <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

We studied the usefulness of flow cytometry for detection of penicillin resistance in E. faecalis and S. aureus by direct binding of commercially available fluorescent penicillin, Bocillin FL, to cells obtained from culture. There were significantly lower percentages of fluorescent cells and median and mean fluorescence values per particle in penicillin-resistant than in penicillin-sensitive strains of both species observed. The method allows rapid detection of penicillin resistance in S. aureus and E. faecalis. The results encourage further investigations on the detection of antibiotic resistance in bacteria using flow cytometry.     

Quantitative study of interactions between <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and <Emphasis Type="Italic">Oenococcus </Emphasis><Emphasis Type="Italic">oeni</Emphasis> strains

This study examines the interactions that occur between Saccharomyces cerevisiae and Oenococcus oeni strains during the process of winemaking. Various yeast/bacteria pairs were studied by applying a sequential fermentation strategy which simulated the natural winemaking process. First, four yeast strains were tested in the presence of one bacterial strain leading to the inhibition of the bacterial component. The extent of inhibition varied widely from one pair to another and closely depended on the specific yeast strain chosen. Inhibition was correlated to weak bacterial growth rather than a reduction in the bacterial malolactic activity. Three of the four yeast strains were then grown with another bacteria strain. Contrary to the first results, this led to the bacterial stimulation, thus highlighting the importance of the bacteria strain. The biochemical profile of the four yeast fermented media exhibited slight variations in ethanol, SO(2) and fatty acids produced as well as assimilable consumed nitrogen. These parameters were not the only factors responsible for the malolactic fermentation inhibition observed with the first bacteria strain. The stimulation of the second has not been reported before in such conditions and remains unexplained.     

Populations of <Emphasis Type="Italic">Xanthomonas citri</Emphasis> pv. <Emphasis Type="Italic">mangiferaeindicae</Emphasis> from Asymptomatic Mango Leaves Are Primarily Endophytic

Terrestrial organic carbon is exported to freshwater systems where it serves as substrate for bacterial growth. Temporal variations in the terrigenous organic carbon support for aquatic bacteria are not well understood. In this paper, we demonstrate how the combined influence of landscape characteristics and hydrology can shape such variations. Using a 13-day bioassay approach, the production and respiration of bacteria were measured in water samples from six small Swedish streams (64° N, 19° E), draining coniferous forests, peat mires, and mixed catchments with typical boreal proportions between forest and mire coverage. Forest drainage supported higher bacterial production and higher bacterial growth efficiency than drainage from mires. The areal export of organic carbon was several times higher from mire than from forest at low runoff, while there was no difference at high flow. As a consequence, mixed streams (catchments including both mire and forest) were dominated by mire organic carbon with low support of bacterial production at low discharge situations but dominated by forest carbon supporting higher bacterial production at high flow. The stimulation of bacterial growth during high-flow episodes was a result of higher relative export of organic carbon via forest drainage rather than increased drainage of specific “high-quality” carbon pools in mire or forest soils.     

Modelling faecal <Emphasis Type="Italic">streptococci</Emphasis> mortality in constructed wetlands implanted with <Emphasis Type="Italic">Eichhornia crassipes</Emphasis>

Faecal streptococci mortality was investigated in a water hyacinth (Eichhornia crassipes) constructed wetland pond. The wetland was 7.5 m long, 1.5 m wide and 1.0 m deep, and was implanted with E. crassipes. In order to assess the performance of the system towards bacterial mortality, a mathematical model, based on plug flow philosophy was developed. The model incorporated the role of factors, namely solar intensity, pH, dissolved oxygen, temperature, sedimentation, and root attached growth. Model analysis strongly suggests that bacterial mortality rate constant was largely influenced by two factors, namely solar intensity and root biofilm attachment, with both contributing approximately 70.5% of removal. The contribution of other factors like temperature, dissolved oxygen, pH and sedimentation on bacterial mortality rate were less significant. For example, dissolved oxygen, pH and sedimentation contributed 5%, 8% and 0.82%, respectively. Thus, the sedimentation factor was omitted from the model because of its insignificant contribution. The same was done for temperature, due to low ambient temperature range (3.1°C) in the study area. The overall model bacterial removal efficiency was 83%.     

Dynamic analysis of <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis> subsp. <Emphasis Type="Italic">bulgaricus</Emphasis> CFL1 physiological characteristics during fermentation

This study aimed at examining and comparing the relevance of various methods in order to discriminate different cellular states of Lactobacillus bulgaricus CFL1 and to improve knowledge on the dynamics of the cellular physiological state during growth and acidification. By using four fluorescent probes combined with multiparametric flow cytometry, membrane integrity, intracellular esterase activity, cellular vitality, membrane depolarization, and intracellular pH were quantified throughout fermentations. Results were compared and correlated with measurements of cultivability, acidification activity (Cinac system), and cellular ability to recover growth in fresh medium (Bioscreen system). The Cinac system and flow cytometry were relevant to distinguish different physiological states throughout growth. Lb. bulgaricus cells maintained their high viability, energetic state, membrane potential, and pH gradient in the late stationary phase, despite the gradual decrease of both cultivability and acidification activity. Viability and membrane integrity were maintained during acidification, at the expense of their cultivability and acidification activity. Finally, this study demonstrated that the physiological state during fermentation was strongly affected by intracellular pH and the pH gradient. The critical pHi of Lb. bulgaricus CFL1 was found to be equal to pH 5.8. Through linear relationships between dpH and cultivability and pHi and acidification activity, pHi and dpH well described the time course of metabolic activity, cultivability, and viability in a single analysis.     

Molecular analysis of the genus <Emphasis Type="Italic">Anoxybacillus</Emphasis> based on sequence similarity of the genes <Emphasis Type="Italic">recN</Emphasis>, <Emphasis Type="Italic">flaA</Emphasis>, and <Emphasis Type="Italic">ftsY</Emphasis>

Genome predictions based on selected genes would be a very welcome approach for taxonomic studies. We analyzed three genes, recN, flaA, and ftsY, for determining if these genes are useful tools for systematic analyses in the genus Anoxybacillus. The genes encoding a DNA repair and genetic recombination protein (recN), the flagellin protein (flaA), and GTPase signal docking protein (ftsY) were sequenced for ten Anoxybacillus species. The sequence comparisons revealed that recN sequence similarities range between 61% and 99% in the genus Anoxybacillus. Comparisons to other bacterial recN genes indicated that levels of similarity did not differ from the levels within genus Anoxybacillus. These data showed that recN is not a useful marker for the genus Anoxybacillus. A 550–600-bp region of the flagellin gene was amplified for all Anoxybacillus strains except for Anoxybacillus contaminans. The sequence similarity of flaA gene varies between 61% and 76%. Comparisons to other bacterial flagellin genes obtained from GenBank (Bacillus, Pectinatus, Proteus, and Vibrio) indicated that the levels of similarity were lower (3–42%). Based on these data, we concluded that the variability in this single gene makes it a particularly useful marker. Another housekeeping gene ftsY suggested to reflect the G+C (mol/mol) content of whole genome was analyzed for Anoxybacillus strains. A mean difference of 1.4% was observed between the G+C content of the gene ftsY and the G+C content of the whole genome. These results showed that the gene ftsY can be used to represent whole G+C content of the Anoxybacillus species.     

Transformation of a filamentous fungus<Emphasis Type="Italic">Cryphonectria parasitica</Emphasis> using<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

AsAgrobacterium tumefaciens, which has long been used to transform plants, is known to transfer T-DNA to budding yeast,Saccharomyces cerevisiae, a variety of fungi were subjected to theA. tumefaciens-mediated transformation to improve their transformation frequency and feasibility. TheA. tumefaciens-mediated transformation of chestnut blight fungus,Cryphonectria parasitica, is performed in this study as the first example of transformation of a hardwood fungal pathogen. The transfer of the binary vector pBIN9-Hg, containing the bacterial hygromycin B phosphotransferase gene under the control of theAspergillus nidulans trpC promoter and terminator, as a selectable marker, led to the selection of more than 1,000 stable, hygromycin B-resistant transformants per 1×10⁶ conidia ofC. parasitica. The putative transformants appeared to be mitotically stable. The transformation efficiency appears to depend on the bacterial strain, age of the bacteria cell culture and ratio of fungal spores to bacterial cells. PCR and Southern blot analysis indicated that the marker gene was inserted at different chromosomal sites. Moreover, three transformants out of ten showed more than two hybridizing bands, suggesting more than two copies of the inserted marker gene are not uncommon.     

Impact of <Emphasis Type="Italic">cry1AC</Emphasis>-carrying <Emphasis Type="Italic">Brassica rapa</Emphasis> subsp. <Emphasis Type="Italic">pekinensis</Emphasis> on leaf bacterial community

The effects of Chinese cabbage (Brassica rapa subsp. pekinensis) carrying cry1AC derived from Bacillus thuringiensis (Bt) on leaf bacterial community were examined by analyzing the horizontal transfer of trans-gene fragments from plants to bacteria. The effect of plant pathogenic bacteria on the gene transfer was also examined using Pseudomonas syringae pathovar. maculicola. The frequency of hygromycin-resistant bacteria did not alter in Bt leaves, though slight increase was observed in Pseudomonas-infected Bt leaves with no statistical significance. The analysis of bacterial community profiles using the denaturing gradient gel electrophoresis (DGGE) fingerprinting indicated that there were slight differences between Bt and control Chinese cabbage, and also that infected tissues were dominated by P. syringae pv. maculicola. However, the cultured bacterial pools were not found to contain any transgene fragments. Thus, no direct evidence of immediate gene transfer from plant to bacteria or acquisition of hygromycin resistance could be observed. Still, long-term monitoring on the possibility of gene transfer is necessary to correctly assess the environmental effects of the Bt crop on bacteria.     

Production of bifunctional single-chain antibody-based fusion proteins in <Emphasis Type="Italic">Pichia </Emphasis><Emphasis Type="Italic">pastoris</Emphasis> supernatants

Recombinant antibody fusion constructs with heterologous functional domains are a promising approach to new therapeutic targeting strategies. However, expression of such constructs is mostly limited to cost and labor-intensive mammalian expression systems. Here we report on the employment of Pichia pastoris for the expression of heterologous antibody fusion constructs with green fluorescent protein, A33scFv::GFP, or with cytosine deaminase, A33scFv::CDy, their production in a biofermenter and a modified purification strategy. Combined, these approaches improved production yields by about thirty times over established standard protocols, with extracellular secretion of the fusion construct reaching 12.0 mg/l. Bifunctional activity of the fusion proteins was demonstrated by flow cytometry and an in-vitro cytotoxicity assay. With equal amounts of purified protein, the modified purification method lead to higher functional results. Our results demonstrate the suitability of methylotrophic Pichia expression systems and laboratory-scale bioreactors for the production of high quantities of bifunctionally active heterologous single-chain fusion proteins.     

Gene gain and loss events in <Emphasis Type="Italic">Rickettsia</Emphasis> and <Emphasis Type="Italic">Orientia</Emphasis>species

Background  

Genome degradation is an ongoing process in all members of the Rickettsiales order, which makes these bacterial species an excellent model for studying reductive evolution through interspecies variation in genome size and gene content. In this study, we evaluated the degree to which gene loss shaped the content of some Rickettsiales genomes. We shed light on the role played by horizontal gene transfers in the genome evolution of Rickettsiales.     

PCR Detection of <Emphasis Type="Italic">Serratia</Emphasis> spp<Emphasis Type="Italic">.</Emphasis> Using Primers Targeting <Emphasis Type="Italic">pfs</Emphasis> and <Emphasis Type="Italic">luxS</Emphasis> Genes Involved in AI-2-Dependent Quorum Sensing

Quorum sensing is the ability of bacteria to communicate and coordinate behavior emitting signaling molecules. A series of primers for PCR detection of Serratia spp. has been designed using as targets the pfs and luxS genes involved in AI-2-dependent quorum sensing. The identities of the PCR products (193 and 102 bp) were confirmed by commercial sequencing. Twenty-seven Serratia strains (representing 10 different species) tested positive for the presence of the pfs and luxS genes, while a total of 7 different species of non-Serratia (25 strains) were tested and gave negative results. The sensitivity and specificity of the pfs- and luxS-based PCR assay were also checked in artificially contaminated bacterial samples. In this study we established a novel method to detect Serratia using quorum-sensing genes as diagnostic markers.     

Quantitative assessment of toxic and nontoxic <Emphasis Type="Italic">Microcystis</Emphasis> colonies in natural environments using fluorescence <Emphasis Type="Italic">in situ</Emphasis> hybridization and flow cytometry

Toxic cyanobacterial blooms constitute a threat to human safety because Microcystis sp. releases microcystins during growth, and particularly during cell death. Therefore, analysis of toxic and nontoxic Microcystis in natural communities is required in order to assess and predict bloom dynamics and toxin production by these organisms. In this study, an analysis combining fluorescence in situ hybridization (FISH) with flow cytometry (FCM) was used to discriminate between toxic and nontoxic Microcystis and also to quantify the percentage of toxic Microcystis present in blooms. The results demonstrate that the combination of FISH and flow cytometry is a useful approach for studying the ecology of Microcystis toxin production and for providing an early warning for toxic Microcystis blooms.     

Somatic hybrids <Emphasis Type="Italic">Solanum nigrum</Emphasis> (+) <Emphasis Type="Italic">S. tuberosum</Emphasis>: morphological assessment and verification of hybridity

Somatic hybrids between the cultivated potato diploid hybrid clone, ZEL-1136, and hexaploid non-tuber-bearing wild species Solanum nigrum L. exhibiting resistance to Phytophthora infestans were regenerated after PEG-mediated fusion of mesophyll protoplasts. The objective was to transfer the late-blight resistance genes from the wild species into plants of the cultivated potato clone. From a total of 59 regenerants, 40 clones survived and have been maintained in vitro on hormone-free MS/2 medium. Thirty-two somatic hybrids were identified by their intermediate morphology (leaves of nigrum type and flowers of tuberosum type) and verified by flow cytometry and random amplified polymorphic DNA (RAPD) patterns. The RAPD analysis of nuclear DNA confirmed the hybrid nature of 29 clones. Flow cytometry revealed a wide range of ploidy in the generated hybrids, from nearly the tetra- to decaploid level. Most of the hybrid clones were stable in vitro, grew vigorously in soil, and set flowers and parthenocarpic berries. However, all of the flowering hybrids were male-sterile. Nine hybrid clones produced tuber-like structures in soil. The most vigorous flowering somatic hybrids were selected for assessment of the late-blight resistance.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of protocorm-like bodies in <Emphasis Type="Italic">Cattleya</Emphasis>

A protocol for in vitro induction of crape myrtle tetraploids using nodes from in vitro-grown shoots (2n = 48) was established. Nodal buds were excised from in vitro-grown shoots, maintained on proliferation medium containing Murashige and Skoog medium supplemented with 4.44 μM 6-benzyladenine , 0.54 μM α-naphthaleneacetic acid, and treated with a range of concentrations of colchicine under three different conditions. Nodal bud explants treated in liquid proliferation medium supplemented with either 15 or 20 mM colchicine for 24 h turned necrotic and died; whereas, those cultured on solid proliferation medium supplemented with either 125 or 250 μM colchicine for 30 days survived, but no tetraploid plants were obtained. However, when explants were cultured in liquid proliferation medium containing 250, 500 or 750 μM colchicine for 10 days, tetraploid plants (2n = 96) were obtained. Incubation of explants in medium containing 750 μM colchicine promoted the highest frequency of survival (40%) of explants and of recovered tetraploids (60%). Morphological and anatomical characteristics of leaves, including leaf index, stomata size and number, stomata index (length/width), and number of chloroplasts in guard cells correlated with ploidy of crape myrtle plants. The number of chloroplasts in guard cells of stomata was a stable and reliable marker in discriminating plants of different ploidy levels. Chromosome counts and flow cytometry confirmed these findings.     

Isolation and screening of <Emphasis Type="Italic">phlD</Emphasis><Superscript>+</Superscript> plant growth promoting rhizobacteria antagonistic to <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis>

Tomato (Lycopersicon esculentum) is important widely grown vegetable in India and its productivity is affected by bacterial wilt disease infection caused by Ralstonia solanacearum. To prevent this disease infection a study was conducted to isolate and screen effective plant growth promoting rhizobacteria (PGPR) antagonistic to R. solanacearum. A total 297 antagonistic bacteria were isolated through dual culture inoculation technique, out of which forty-two antagonistic bacteria were found positive for phlD gene by PCR amplification using two primer sets Phl2a:Phl2b and B2BF:BPR4. The genetic diversity of phlD ⁺ bacteria was studied by amplified 16S rDNA restriction analysis and demonstrated eleven groups at 65% similarity level. Out of these 42 phlD ⁺ antagonistic isolates, twenty exhibited significantly fair plant growth promoting activities like phosphate solubilization (0.92–5.33%), 25 produced indole acetic acid (1.63–7.78 μg ml⁻¹) and few strains show production of antifungal metabolites (HCN and siderophore). The screening of PGPR (phlD ⁺) for suppression of bacterial wilt disease in glass house conditions was showed ten isolated phlD ⁺ bacteria were able to suppress infection of bacterial wilt disease in tomato plant (var. Arka vikas) in the presence R. solanacearum. The PGPR (phlD ⁺) isolates s188, s215 and s288 was observed to be effective plant growth promoter as it shows highest dry weight per plant (3.86, 3.85 and 3.69 g plant⁻¹ respectively). The complete absence of wilt disease symptoms in tomato crop plants was observed by these treatments compared to negative control. Therefore inoculation of tomato plant with phlD ⁺ isolate s188 and other similar biocontrol agents may prove to be a positive strategy for checking wilt disease and thus improving plant vigor.