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A Burkholderia pseudomallei Type III Secreted Protein, BopE, Facilitates Bacterial Invasion of Epithelial Cells and Exhibits Guanine Nucleotide Exchange Factor Activity. Mark P. Stevens, 2003.We report the characterization of BopE, a type III secreted protein that is encoded adjacent to the Burkholderia pseudomallei bsa locus and is homologous to Salmonella enterica SopE/SopE2 . Inactivation of bopE impaired bacterial entry into HeLa cells, indicating that BopE facilitates invasion . Consistent with this notion, BopE expressed in eukaryotic cells induced rearrangements in the subcortical actin cytoskeleton, and purified BopE exhibited guanine nucleotide exchange factor activity for Cdc42 and Rac1 in vitro . Whole-Genome Sequence Variation among Multiple Isolates of Pseudomonas aeruginosa. David H. Spencer, 2003.Whole-genome shotgun sequencing was used to study the sequence variation of three Pseudomonas aeruginosa isolates, two from clonal infections of cystic fibrosis patients and one from an aquatic environment, relative to the genomic sequence of reference strain PAO1 . The majority of the PAO1 genome is represented in these strains; however, at least three prominent islands of PAO1-specific sequence are apparent . Conversely,  10% of the sequencing reads derived from each isolate fail to align with the PAO1 backbone . While average sequence variation among all strains is roughly 0.5%, regions of pronounced differences were evident in whole-genome scans of nucleotide diversity . We analyzed two such divergent loci, the pyoverdine and O-antigen biosynthesis regions, by complete resequencing . A thorough analysis of isolates collected over time from one of the cystic fibrosis patients revealed independent mutations resulting in the loss of O-antigen synthesis alternating with a mucoid phenotype . Overall, we conclude that most of the PAO1 genome represents a core P . aeruginosa backbone sequence while the strains addressed in this study possess additional genetic material that accounts for at least 10% of their genomes . Approximately half of these additional sequences are novel .
Development of Sinorhizobium meliloti Pilot Macroarrays for Transcriptome Analysis. Hélène Bergès, 2003.In order to prepare for whole-genome expression analysis in Sinorhizobium meliloti, pilot DNA macroarrays were designed for 34 genes of known regulation . The experimental parameters assessed were the length of the PCR products, the influence of a tag at the 5' end of the primers, and the method of RNA labeling . Variance and principal-component analysis showed that the most important nonbiological parameter was the labeling method . The sizes of PCR products were also found to be important, whereas the influence of 5' tags was minimal . The variability between replicated spots on a membrane was found to be low . These experimental procedures were validated by analyzing the effects of microaerobic conditions on gene expression . Physicochemical Conditions and Microbial Activities in the Highly Alkaline Gut of the Humus-Feeding Larva of Pachnoda ephippiata (Coleoptera: Scarabaeidae). Thorsten Lemke, 2003.The soil macrofauna plays an important role in the carbon and nitrogen cycle of terrestrial ecosystems . In order to gain more insight into the role of the intestinal microbiota in transformation and mineralization of organic matter during gut passage, we characterized the physicochemical conditions, microbial activities, and community structure in the gut of our model organism, the humus-feeding larva of the cetoniid beetle Pachnoda ephippiata . Microsensor measurements revealed an extreme alkalinity in the midgut, with highest values (pH > 10) between the second and third crown of midgut ceca . Both midgut and hindgut were largely anoxic, but despite the high pH, the redox potential of the midgut content was surprisingly high even in the largest instar . However, reducing conditions prevailed in the hindgut paunch of all instars (Eh  -100 mV) . Both gut compartments possessed a pronounced gut microbiota, with highest numbers in the hindgut, and microbial fermentation products were present in high concentrations . The stimulation of hindgut methanogenesis by exogenous electron donors, such as H2, formate, and methanol, together with considerable concentrations of formate in midgut and hemolymph, suggests that midgut fermentations are coupled to methanogenesis in the hindgut by an intercompartmental transfer of reducing equivalents via the hemolymph . The results of a cultivation-based enumeration of the major metabolic groups in midgut and hindgut, which yielded high titers of lactogenic, propionigenic, and acetogenic bacteria, are in good agreement not only with the accumulation of microbial fermentation products in the respective compartments but also with the results of a cultivation-independent characterization of the bacterial communities reported in the companion paper (M . Egert, B . Wagner, T . Lemke, A . Brune, and M . W . Friedrich, Appl . Environ . Microbiol . 69:6659-6668, 2003) .
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