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Early Colonization Events in the Mutualistic Association between Steinernema carpocapsae Nematodes and Xenorhabdus nematophila Bacteria. Eric C. Martens, 2003.The bacterium Xenorhabdus nematophila is a mutualist of the entomopathogenic nematode Steinernema carpocapsae . During its life cycle, the bacterium exists both separately from the nematode and as an intestinal resident of a nonfeeding nematode form, the infective juvenile (IJ) . The progression of X . nematophila from an ex vivo existence to a specific and persistent colonization of IJs is a model to understand the mechanisms mediating the initiation and maintenance of benign host-microbe interactions . To help characterize this process, we constructed an X . nematophila strain that constitutively expresses green fluorescent protein, which allowed its presence to be monitored within IJs . Using this strain, we showed that few bacterial cells initiate colonization of an individual IJ and that these grow inside the lumen of the IJ intestine in a reproducible polyphasic pattern during colonization . In accordance with these two observations, we demonstrated that the final population of bacteria in a nematode is of predominantly monoclonal origin, suggesting that only one or two bacterial clones initiate or persist during colonization of an individual nematode . These data suggest that X . nematophila initiates IJ colonization by competing for limited colonization sites or resources within the nematode intestine . This report represents the first description of the biological interactions occurring between X . nematophila and S . carpocapsae during the early stages of the colonization process, provides insights into the physiology of X . nematophila in its host niche, and will facilitate interpretation of future data regarding the molecular events mediating this process . Diversity and Seasonal Changes of Uncultured Planctomycetales in River Biofilms. I. H. M. Brümmer, 2004.Cell counts of planctomycetes showed that there were high levels of these organisms in the summer and low levels in the winter in biofilms grown in situ in two polluted rivers, the Elbe River and the Spittelwasser River . In this study 16S rRNA-based methods were used to investigate if these changes were correlated with changes in the species composition . Planctomycete-specific clone libraries of the 16S rRNA genes found in both rivers showed that there were seven clusters, which were distantly related to the genera Pirellula, Planctomyces, and Gemmata . The majority of the sequences from the Spittelwasser River were affiliated with a cluster related to Pirellula, while the majority of the clones from the Elbe River fell into three clusters related to Planctomyces and one deeply branching cluster related to Pirellula . Some clusters also contained sequences derived from freshwater environments worldwide, and the similarities to our biofilm clones were as high as 99.8%, indicating the presence of globally distributed freshwater clusters of planctomycetes that have not been cultivated yet . Community fingerprints of planctomycete 16S rRNA genes were generated by temperature gradient gel electrophoresis from Elbe River biofilm samples collected monthly for 1 year . Sixteen bands were identified, and for the most part these bands represented organisms related to the genus Planctomyces. The fingerprints showed that there was strong seasonality of most bands and that there were clear differences in the summer and the winter . Thus, seasonal changes in the abundance of Planctomycetales in river biofilms were coupled to shifts in the community composition . Effects of the Chromosome Partitioning Protein Spo0J (ParB) on oriC Positioning and Replication Initiation in Bacillus subtilis. Philina S. Lee, 2003.Spo0J (ParB) of Bacillus subtilis is a DNA-binding protein that belongs to a conserved family of proteins required for efficient plasmid and chromosome partitioning in many bacterial species . We found that Spo0J contributes to the positioning of the chromosomal oriC region, but probably not by recruiting the origin regions to specific subcellular locations . In wild-type cells during exponential growth, duplicated origin regions were generally positioned around the cell quarters . In a spo0J null mutant, sister origin regions were often closer together, nearer to midcell . We found, by using a Spo0J-green fluorescent protein [GFP] fusion, that the subcellular location of Spo0J was a consequence of the chromosomal positions of the Spo0J binding sites . When an array of binding sites (parS sites) were inserted at various chromosomal locations in the absence of six of the eight known parS sites, Spo0J-GFP was no longer found predominantly at the cell quarters, indicating that Spo0J is not sufficient to recruit chromosomal parS sites to the cell quarters . spo0J also affected chromosome positioning during sporulation . A spo0J null mutant showed an increase in the number of cells with some origin-distal regions located in the forespore . In addition, a spo0J null mutation caused an increase in the number of foci per cell of LacI-GFP bound to arrays of lac operators inserted in various positions in the chromosome, including the origin region, an increase in the DNA-protein ratio, and an increase in origins per cell, as determined by flow cytometry . These results indicate that the spo0J mutant produced a significant proportion of cells with increased chromosome content, probably due to increased and asynchronous initiation of DNA replication . Influence of Surface Characteristics on the Stability of Cryptosporidium parvum Oocysts. Michael A. Butkus, 2003.Microelectrophoresis is a common technique for probing the surface chemistry of the Cryptosporidium parvum oocyst . Results of previous studies of the electrophoretic mobility of C . parvum oocysts in which microelectrophoresis was used are incongruent . In this work we demonstrated that capillary electrophoresis may also be used to probe the surface characteristics of C . parvum oocysts, and we related the surface chemistry of C . parvum oocysts to their stability in water . Capillary electrophoresis results indicated that oocysts which were washed in a phosphate buffer solution had neutrally charged surfaces . Inactivation of oocysts with formalin did not influence their electrophoretic mobility, while oocyst populations that were washed in distilled water consisted of cells with both neutral and negative surface charges . These results indicate that washing oocysts in low-ionic-strength distilled water can impart a negative charge to a fraction of the oocysts in the sample . Rapid coagulation experiments indicated that oocysts did not aggregate in a 0.5 M NaCl solution; oocyst stability in the salt solution may have been the result of Lewis acid-base forces, steric stabilization, or some other factor . The presence of sucrose and Percoll could not be readily identified on the surface of C . parvum oocysts by attenuated total reflectance-Fourier transform infrared spectroscopy, suggesting that these purification reagents may not be responsible for the stability of the uncharged oocysts . These findings imply that precipitate enmeshment may be the optimal mechanism of coagulation for removal of oocysts in water treatment systems . The results of this work may help elucidate the causes of variation in oocyst surface characteristics, may ultimately lead to improved removal efficiencies in full-scale water treatment systems, and may improve fate and transport predictions for oocysts in natural systems . Microbial Community Structure in Midgut and Hindgut of the Humus-Feeding Larva of Pachnoda ephippiata (Coleoptera: Scarabaeidae). Markus Egert, 2003.The guts of soil-feeding macroinvertebrates contain a complex microbial community that is involved in the transformation of ingested soil organic matter . In a companion paper (T . Lemke, U . Stingl, M . Egert, M . W . Friedrich, and A . Brune, Appl . Environ . Microbiol . 69:6650-6658, 2003), we show that the gut of our model organism, the humivorous larva of the cetoniid beetle Pachnoda ephippiata, is characterized by strong midgut alkalinity, high concentrations of microbial fermentation products, and the presence of a diverse, yet unstudied microbial community . Here, we report on the community structure of bacteria and archaea in the midgut, hindgut, and food soil of P . ephippiata larvae, determined with cultivation-independent techniques . Clone libraries and terminal restriction fragment length polymorphism analysis of 16S rRNA genes revealed that the intestines of P . ephippiata larvae contain a complex gut microbiota that differs markedly between midgut and hindgut and that is clearly distinct from the microbiota in the food soil . The bacterial community is dominated by phylogenetic groups with a fermentative metabolism (Lactobacillales, Clostridiales, Bacillales, and Cytophaga-Flavobacterium-Bacteroides [CFB] phylum), which is corroborated by high lactate and acetate concentrations in the midgut and hindgut and by the large numbers of lactogenic and acetogenic bacteria in both gut compartments reported in the companion paper . Based on 16S rRNA gene frequencies, Actinobacteria dominate the alkaline midgut, while the hindgut is dominated by members of the CFB phylum . The archaeal community, however, is less diverse . 16S rRNA genes affiliated with mesophilic Crenarchaeota, probably stemming from the ingested soil, were most frequent in the midgut, whereas Methanobacteriaceae-related 16S rRNA genes were most frequent in the hindgut . These findings agree with the reported restriction of methanogenesis to the hindgut of Pachnoda larvae .
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