Analysis of outer membrane vesicle associated proteins isolated from the plant pathogenic bacterium <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. campestris

Background  

Outer membrane vesicles (OMVs) are released from the outer membrane of many Gram-negative bacteria. These extracellular compartments are known to transport compounds involved in cell-cell signalling as well as virulence associated proteins, e.g. the cytolysine from enterotoxic E. coli.     

Complete genome of the mutualistic, N2-fixing grass endophyte Azoarcus sp. strain BH72

Azoarcus sp. strain BH72, a mutualistic endophyte of rice and other grasses, is of agrobiotechnological interest because it supplies biologically fixed nitrogen to its host and colonizes plants in remarkably high numbers without eliciting disease symptoms. The complete genome sequence is 4,376,040-bp long and contains 3,992 predicted protein-coding sequences. Genome comparison with the Azoarcus-related soil bacterium strain EbN1 revealed a surprisingly low degree of synteny. Coding sequences involved in the synthesis of surface components potentially important for plant-microbe interactions were more closely related to those of plant-associated bacteria. Strain BH72 appears to be 'disarmed' compared to plant pathogens, having only a few enzymes that degrade plant cell walls; it lacks type III and IV secretion systems, related toxins and an N-acyl homoserine lactones-based communication system. The genome contains remarkably few mobile elements, indicating a low rate of recent gene transfer that is presumably due to adaptation to a stable, low-stress microenvironment.     

Genome sequence of the ubiquitous hydrocarbon-degrading marine bacterium Alcanivorax borkumensis

Alcanivorax borkumensis is a cosmopolitan marine bacterium that uses oil hydrocarbons as its exclusive source of carbon and energy. Although barely detectable in unpolluted environments, A. borkumensis becomes the dominant microbe in oil-polluted waters. A. borkumensis SK2 has a streamlined genome with a paucity of mobile genetic elements and energy generation-related genes, but with a plethora of genes accounting for its wide hydrocarbon substrate range and efficient oil-degradation capabilities. The genome further specifies systems for scavenging of nutrients, particularly organic and inorganic nitrogen and oligo-elements, biofilm formation at the oil-water interface, biosurfactant production and niche-specific stress responses. The unique combination of these features provides A. borkumensis SK2 with a competitive edge in oil-polluted environments. This genome sequence provides the basis for the future design of strategies to mitigate the ecological damage caused by oil spills.     

Unusual outer membrane lipid composition of the gram-negative, lipopolysaccharide-lacking myxobacterium Sorangium cellulosum So ce56

The gram-negative myxobacterium Sorangium cellulosum So ce56 bears the largest bacterial genome published so far, coding for nearly 10,000 genes. Careful analysis of this genome data revealed that part of the genes coding for the very well conserved biosynthesis of lipopolysaccharides (LPS) are missing in this microbe. Biochemical analysis gave no evidence for the presence of LPS in the membranes of So ce56. By analyzing the lipid composition of its outer membrane sphingolipids were identified as the major lipid class, together with ornithine-containing lipids (OL) and ether lipids. A detailed analysis of these lipids resulted in the identification of more than 50 structural variants within these three classes, which possessed several interesting properties regarding to LPS replacement, mediators in myxobacterial differentiation, as well as potential bioactive properties. The sphingolipids with the basic structure C9-methyl-C(20)-sphingosine possessed as an unusual trait C9-methylation, which is common to fungi but highly uncommon to bacteria. Such sphingolipids have not been found in bacteria before, and they may have a function in myxobacterial development. The OL, also identified in myxobacteria for the first time, contained acyloxyacyl groups, which are also characteristic for LPS and might replace those in certain functions. Finally, the ether lipids may serve as biomarkers in myxobacterial development.     

Insights into the genome of the xanthan-producing phytopathogen Xanthomonas arboricola pv. pruni 109 by comparative genomic hybridization

The phytopathogenic bacterium Xanthomonas arboricola pv. pruni is the causal agent of Prunus Bacterial Spot disease that infects cultivated Prunus species and their hybrids. Furthermore, X. arboricola pv. pruni (Xap) plays a role in biotechnology since it produces xanthan gum, an important biopolymer used mainly in the food, oil, and cosmetics industry. To gain first insights into the genome composition of this pathovar, genomic DNA of X. arboricola pv. pruni strains was compared to the genomes of reference strains X. campestris pv. campestris B100 (Xcc B100) and X. campestris pv. vesicatoria 85-10 (Xcv 85-10) applying microarray-based comparative genomic hybridizations (CGH). The results implied that X. arboricola pv. pruni 109 lacks 6.67% and 5.21% of the genes present in the reference strains Xcc B100 and Xcv 85-10, respectively. Most of the missing genes were found to be organized in clusters and do not belong to the core genome of the two reference strains. Often they encode mobile genetic elements. Furthermore, the absence of gene clusters coding for the lipopolysaccharide (LPS) O-antigens of Xcc B100 and Xcv 85-10 indicates that the structure of the O-antigen of X. arboricola pv. pruni 109 differs from that of Xcc B100 and Xcv 85-10.     

The ubiquitous plasmid pXap41 in the invasive phytopathogen Xanthomonas arboricola pv. pruni: complete sequence and comparative genomic analysis

The complete DNA sequence of the 41 102-bp plasmid pXap41 from the invasive plant pathogen Xanthomonas arboricola pv. pruni CFBP 5530 was determined and its 44 coding regions were annotated. Comparative analysis with 15 Xanthomonas plasmids and 19 complete genomes revealed that nearly one-fourth of this plasmid has high sequence identity to plasmid pXAC64 and an 8.8-kb chromosomal region of Xanthomonas axonopodis pv. citri strain 306 carrying genes that encode type III effectors and helper proteins. The presence of pXap41 in all X. arboricola pv. pruni genotypes was confirmed for eight strains by plasmid profiling and for 35 X. arboricola pv. pruni isolates with a new plasmid multiplex PCR assay. This plasmid was not detected in any other X. arboricola pathovars (n=12), indicating the potential for the application of the pXap41 PCR method as a pathovar-level detection and identification tool.     

Impact of C60 Adsorption on Surface Plasmon Polaritons on Self-Assembled Ag(111) Islands on Si(111)

The influence of C₆₀ adsorption on the properties of surface plasmon polaritons on small Ag islands is discussed. Under illumination with UV light as well as under illumination with femtosecond laser pulses, a decrease of the photoemission yield with increasing C₆₀ coverage is observed. With angular resolved measurements, changes of the band structure during deposition are studied. Based on these experiments, an increase of the work function with increasing coverage is measured. In two photon photoemission, the surface plasmons are imaged as a periodic moiré pattern, the wavelength of which changes because of a modified effective surface dielectric function. Our findings imply that the wavelength of the plasmon wave becomes shorter as a result. Finally, a decrease of the intensity of the moiré pattern maxima compared with the intensity of the first maximum with increasing C₆₀ coverage has been observed. Accordingly, the damping of the plasmon wave becomes stronger.     

Identification of Xanthomonas campestris pv. campestris galactose utilization genes from transcriptome data

A 70 mer oligonucleotide microarray was constructed to analyze genome-wide expression profiles of Xanthomonas campestris pv. campestris B100, a plant-pathogenic bacterium that is industrially employed to produce the exopolysaccharide xanthan gum which has many applications as a stabilizing, thickening, gelling, and emulsifying agent in food, pharmaceutical, and cosmetic industries. As an application example, global changes of gene expression were monitored during growth of X. campestris pv. campestris B100 on two different carbon sources. Exponential growing bacterial cultures were incubated either for 1h or permanently in minimal medium supplemented with 1% galactose in comparison to growth in minimal medium supplemented with 1% glucose. Six genes were identified that were significantly increased in gene expression under both growth conditions. These genes were located in three distinguished chromosomal regions in operon-like gene clusters. Genes from these clusters encode secreted glycosidases, which were predicted to be specific for galactose-containing carbohydrates, as well as transport proteins probably located in the outer and inner cell membrane. Finally genes from one cluster code for cytoplasmic enzymes of a metabolic pathway specific for the breakdown of galactose to intermediates of glycolysis.     

Application of a Short Intracellular pH Method to Flow Cytometry for Determining Saccharomyces cerevisiae Vitality

The measurement of yeast''s intracellular pH (ICP) is a proven method for determining yeast vitality. Vitality describes the condition or health of viable cells as opposed to viability, which defines living versus dead cells. In contrast to fluorescence photometric measurements, which show only average ICP values of a population, flow cytometry allows the presentation of an ICP distribution. By examining six repeated propagations with three separate growth phases (lag, exponential, and stationary), the ICP method previously established for photometry was transferred successfully to flow cytometry by using the pH-dependent fluorescent probe 5,6-carboxyfluorescein. The correlation between the two methods was good (r² = 0.898, n = 18). With both methods it is possible to track the course of growth phases. Although photometry did not yield significant differences between exponentially and stationary phases (P = 0.433), ICP via flow cytometry did (P = 0.012). Yeast in an exponential phase has a unimodal ICP distribution, reflective of a homogeneous population; however, yeast in a stationary phase displays a broader ICP distribution, and subpopulations could be defined by using the flow cytometry method. In conclusion, flow cytometry yielded specific evidence of the heterogeneity in vitality of a yeast population as measured via ICP. In contrast to photometry, flow cytometry increases information about the yeast population''s vitality via a short measurement, which is suitable for routine analysis.Yeast plays an important role in the food industry. It is primarily used for making bread, beer, and wine, and the flavor and aromatic compounds it produces are characteristic of these fermented products. The physiological state of the yeast biomass influences the fermentation performance and thus the quality of the resulting product, e.g., of beer (1, 32). Food processors who depend on yeast health for consistent fermentations demand a method to measure yeast''s physiological condition during yeast growth, that is, its vitality. The term “vitality” refers to the health of living biomass where high vitality results in a fast fermentation with minimal undesired by-products, while low vitality results in sluggish or poorly attenuating fermentation. In contrast, the term “viability” only distinguishes between dead and alive cells.Several methods exist to measure yeast vitality such as carbon dioxide production (10), vicinal diketone reduction (3), glycogen and trehalose staining (16-18), vital titration (30), measurement of the specific oxygen uptake rate (27, 38), and the acidification power test (12, 22). One important method for vitality measurement is the detection of intracellular pH (ICP) (9, 19, 20, 31).Intracellular metabolic reactions in yeast are catalyzed by enzymes that have their optimum working pH in a neutral range (23). During fermentation or propagation, yeast produces carbon dioxide and organic acids and releases protons to the medium. To maintain a desired ICP, yeast continuously pumps protons against a gradient from the cytosol to the extracellular medium. The regulating proton pump is plasma-membrane-ATPase (4). The transmembrane proton gradient has to be sustained since the transport of important nutrients such as maltose or amino acids depends on it. The efficiency of glycolysis and gluconeogenesis is affected by ICP. The higher the proton extrusion rate, the more active is the metabolism. Thus, a high ICP indicates high yeast vitality. Yeast with the highest ICP ferment the best (1).Techniques using fluorescence photometry and the pH-dependent dye, 5,6-carboxyfluorescein (CF), for measuring yeast ICP have been published (1, 20, 21, 33). These proved reliable but only yield an average value of ICP of a yeast population. Based on the method of Imai (21), a shorter method for measuring ICP was developed by Thiele and Back (33) using photometry whereby the sample preparation time was reduced from approximately 3.5 to 1.25 h. This reduction made it suitable for routine analysis.In the present study and according to Thiele and Back (33), the nonfluorescent, esterified form of CF, 5,6-carboxyfluorescein-diacetate (CF-DA), was used for cell loading because it is more capable of passing through cell membranes than its nonesterified counterpart (6). After the cells were infused with CF-DA, yeast esterase enzymes split the molecule into acetate residues and the actual fluorescent probe CF. Excitation with a certain wavelength results in different fluorescent intensities that depend on both the pH of the cell cytoplasm and the dye concentration. In order to receive results independent on probe concentration, a ratio between pH-dependent and pH-independent fluorescence intensities of the sample can be used to produce data depending on pH only (20, 35).In addition to measuring ICP via fluorescence photometry, flow cytometry can also serve as detection system of CF fluorescent intensities corresponding to ICP. Flow cytometry is becoming a standard tool in biotechnology research and has been successfully applied to analyze and optimize yeast processes (13, 14, 15, 24, 36). Cytometric measurements provide the opportunity to detect single cells. With this method it is possible to describe a whole yeast population in detail by illustrating the distribution of ICP (35, 39). In contrast, photometry only reflects an average value of the population.We compare here the application of the short ICP method executed via fluorescence photometry to flow cytometry. During the propagation of brewer''s yeast, three different growth phases (the lag, exponential, and stationary phases) were compared to determine whether the vitality varied during growth and whether this variation was detectable by both methods. Since flow cytometry offers the opportunity to identify subpopulations (35, 36), we hypothesized that it was possible to detect yeast subpopulations with differing ICP during propagation.