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Interaction between Protein Subunits of the Type IV Secretion System of Bartonella henselae. Alireza Shamaei-Tousi, 2004.In this study we used the yeast two-hybrid system to identify interactions between protein subunits of the virB type IV secretion system of Bartonella henselae . We report interactions between inner membrane and periplasmic proteins, the pilus polypeptide, and the core complex and a novel interaction between VirB3 and VirB5 . Isolation, Characterization, and Identification of Bacterial Contaminants in Semifinal Gelatin Extracts. E. De Clerck, 2004.Bacterial contamination of gelatin is of great concern . Indeed, this animal colloid has many industrial applications, mainly in food and pharmaceutical products . In a previous study (E . De Clerck and P . De Vos, Syst . Appl . Microbiol . 25:611-618), contamination of a gelatin production process with a variety of gram-positive and gram-negative bacteria was demonstrated . In this study, bacterial contamination of semifinal gelatin extracts from several production plants was examined . Since these extracts are subjected to harsh conditions during production and a final ultrahigh-temperature treatment, the bacterial load at this stage is expected to be greatly reduced . In total, 1,129 isolates were obtained from a total of 73 gelatin batches originating from six different production plants . Each of these batches was suspected of having bacterial contamination based on quality control testing at the production plant from which it originated . For characterization and identification of the 1,129 bacterial isolates, repetitive-element PCR was used to obtain manageable groups . Representative strains were identified by means of 16S rRNA genesequencing, species-specific gyrB PCR, and gyrA and rpoB sequencing and were tested for gelatinase activity . The majority of isolates belonged to members of Bacillus or related endospore-forming genera . Representative strains were identified as Bacillus cereus, Bacillus coagulans, Bacillus fumarioli, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, Bacillus sonorensis, Bacillus subtilis, Bacillus gelatini, Bacillus thermoamylovorans, Anoxybacillus contaminans, Anoxybacillus flavithermus, Brevibacillus agri, Brevibacillus borstelensis, and Geobacillus stearothermophilus . The majority of these species include strains exhibiting gelatinase activity . Moreover, some of these species have known pathogenic properties . These findings are of great concern with regard to the safety and quality of gelatin and its applications . Examination of the Borrelia burgdorferi Transcriptome in Ixodes scapularis during Feeding. Sukanya Narasimhan, 2002.Borrelia burgdorferi gene expression within the guts of engorging Ixodes scapularis ticks was examined by use of differential immunoscreening and differential expression with a customized amplified library . Fourteen chromosomal genes involved in energy metabolism, substrate transport, and signal transduction and 10 (4 chromosomal and 6 plasmid) genes encoding putative lipoproteins and periplasmic proteins were preferentially expressed in engorging ticks . These data demonstrate a new approach to the global analysis of B . burgdorferi genes that are preferentially expressed within the vector during feeding . A Single Nucleotide Exchange in the wzy Gene Is Responsible for the Semirough O6 Lipopolysaccharide Phenotype and Serum Sensitivity of Escherichia coli Strain Nissle 1917. Lubomir Grozdanov, 2002.Structural analysis of lipopolysaccharide (LPS) isolated from semirough, serum-sensitive Escherichia coli strain Nissle 1917 (DSM 6601, serotype O6:K5:H1) revealed that this strain's LPS contains a bisphosphorylated hexaacyl lipid A and a tetradecasaccharide consisting of one E . coli O6 antigen repeating unit attached to the R1-type core . Configuration of the GlcNAc glycosidic linkage between O-antigen oligosaccharide and core (ß) differs from that interlinking the repeating units in the E . coli O6 antigen polysaccharide (  ) . The wa* and wb* gene clusters of strain Nissle 1917, required for LPS core and O6 repeating unit biosyntheses, were subcloned and sequenced . The DNA sequence of the wa* determinant (11.8 kb) shows 97% identity to other R1 core type-specific wa* gene clusters . The DNA sequence of the wb* gene cluster (11 kb) exhibits no homology to known DNA sequences except manC and manB . Comparison of the genetic structures of the wb*O6 (wb* from serotype O6) determinants of strain Nissle 1917 and of smooth and serum-resistant uropathogenic E . coli O6 strain 536 demonstrated that the putative open reading frame encoding the O-antigen polymerase Wzy of strain Nissle 1917 was truncated due to a point mutation . Complementation with a functional wzy copy of E . coli strain 536 confirmed that the semirough phenotype of strain Nissle 1917 is due to the nonfunctional wzy gene . Expression of a functional wzy gene in E . coli strain Nissle 1917 increased its ability to withstand antibacterial defense mechanisms of blood serum . These results underline the importance of LPS for serum resistance or sensitivity of E . coli .
Recognition of DNA by Three Ferric Uptake Regulator (Fur) Homologs in Bacillus subtilis. Mayuree Fuangthong, 2003.Bacillus subtilis contains three Fur homologs: Fur, PerR, and Zur . Despite significant sequence similarities, they respond to different stimuli and regulate different sets of genes . DNA target site comparisons indicate that all three paralogs recognize operators with a core 7-1-7 inverted repeat . The corresponding consensus sequences are identical at five or more of the seven defined positions . Using site-directed mutagenesis, the Per box at the mrgA promoter was altered to mimic the core 7-1-7 motif of the Fur and Zur boxes . In vitro, the mrgA promoter containing a Zur box was only recognized by Zur, as demonstrated by DNase I footprinting assays . In contrast, both Fur and PerR bound to the mrgA promoter region containing a consensus Fur box . Expression analysis of these promoters is consistent with the in vitro data demonstrating as few as 1 or 2 base changes per half-site are sufficient to alter regulation . Similarly, the Fur box at the feuA promoter can be converted into a Per or a Zur box by appropriate mutations . While both Fur and PerR could recognize some of the same synthetic operator sequences, no naturally occurring sites are known that are subject to dual regulation . However, the PerR-regulated zosA gene is controlled from a regulatory region that contains both Per and Fur boxes . Although purified Fur protein bound to the candidate Fur boxes, Fur has little effect on zosA expression—possibly due to the location of the Fur boxes relative to the zosA promoter . Together, our results identify two nucleotide positions that are important for the ability of PerR, Fur, and Zur to distinguish among the many closely related operator sites present in the B . subtilis genome . Heat Shock Response by the Hyperthermophilic Archaeon Pyrococcus furiosus. Keith R. Shockley, 2003.Collective transcriptional analysis of heat shock response in the hyperthermophilic archaeon Pyrococcus furiosus was examined by using a targeted cDNA microarray in conjunction with Northern analyses . Differential gene expression suggests that P . furiosus relies on a cooperative strategy of rescue (thermosome [Hsp60], small heat shock protein [Hsp20], and two VAT-related chaperones), proteolysis (proteasome), and stabilization (compatible solute formation) to cope with polypeptide processing during thermal stress .
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