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Analysis of the Genome Structure of the Nonpathogenic Probiotic Escherichia coli Strain Nissle 1917. Lubomir Grozdanov, 2004.Nonpathogenic Escherichia coli strain Nissle 1917 (O6:K5:H1) is used as a probiotic agent in medicine, mainly for the treatment of various gastroenterological diseases . To gain insight on the genetic level into its properties of colonization and commensalism, this strain's genome structure has been analyzed by three approaches: (i) sequence context screening of tRNA genes as a potential indication of chromosomal integration of horizontally acquired DNA, (ii) sequence analysis of 280 kb of genomic islands (GEIs) coding for important fitness factors, and (iii) comparison of Nissle 1917 genome content with that of other E . coli strains by DNA-DNA hybridization . PCR-based screening of 324 nonpathogenic and pathogenic E . coli isolates of different origins revealed that some chromosomal regions are frequently detectable in nonpathogenic E . coli and also among extraintestinal and intestinal pathogenic strains . Many known fitness factor determinants of strain Nissle 1917 are localized on four GEIs which have been partially sequenced and analyzed . Comparison of these data with the available knowledge of the genome structure of E . coli K-12 strain MG1655 and of uropathogenic E . coli O6 strains CFT073 and 536 revealed structural similarities on the genomic level, especially between the E . coli O6 strains . The lack of defined virulence factors (i.e., alpha-hemolysin, P-fimbrial adhesins, and the semirough lipopolysaccharide phenotype) combined with the expression of fitness factors such as microcins, different iron uptake systems, adhesins, and proteases, which may support its survival and successful colonization of the human gut, most likely contributes to the probiotic character of E . coli strain Nissle 1917 . Secretion of Virulence Proteins from Campylobacter jejuni Is Dependent on a Functional Flagellar Export Apparatus. Michael E. Konkel, 2004.Campylobacter jejuni, a gram-negative motile bacterium, secretes a set of proteins termed the Campylobacter invasion antigens (Cia proteins) . The purpose of this study was to determine whether the flagellar apparatus serves as the export apparatus for the Cia proteins . Mutations were generated in five genes encoding three structural components of the flagella, the flagellar basal body (flgB and flgC), hook (flgE2), and filament (flaA and flaB) genes, as well as in genes whose products are essential for flagellar protein export (flhB and fliI) . While mutations that affected filament assembly were found to be nonmotile (Mot–) and did not secrete Cia proteins (S–), a flaA (flaB+) filament mutant was found to be nonmotile but Cia protein secretion competent (Mot–, S+) . Complementation of a flaA flaB double mutant with a shuttle plasmid harboring either the flaA or flaB gene restored Cia protein secretion, suggesting that Cia export requires at least one of the two filament proteins . Infection of INT 407 human intestinal cells with the C . jejuni mutants revealed that maximal invasion of the epithelial cells required motile bacteria that are secretion competent . Collectively, these data suggest that the C . jejuni Cia proteins are secreted from the flagellar export apparatus . Structure and Function of Hib Pili from Haemophilus influenzae Type b. Xiang-Qi Mu, 2002.Pathogenic bacteria are specifically adapted to bind to their customary host . Disease is then caused by subsequent colonization and/or invasion of the local environmental niche . Initial binding of Haemophilus influenzae type b to the human nasopharynx is facilitated by Hib pili, filaments expressed on the bacterial surface . With three-dimensional reconstruction of electron micrograph images, we show that Hib pili comprise a helix 70 Å in diameter with threefold symmetry . The Hib pilus filament has 3.0 subunits per turn, with each set of three subunits translated 26.9 Å along and rotated 53 degrees about the helical axis . Amino acid sequence analysis of pilins from Hib pili and from P-pili expressed on uropathogenic Escherichia coli were used to predict the physical location of the highly variable and immunogenic region of the HifA pilin in the Hib pilus structure . Structural differences between Hib pili and P-pili suggest a difference in the strategies by which bacteria remain bound to their host cells: P-pili were shown to be capable of unwinding to five times their original length (E . Bullitt and L . Makowski, Nature 373:164-167, 1995), while damage to Hib pili occurs by slight shearing of subunits with respect to those further along the helical axis . This capacity to resist unwinding may be important for continued adherence of H . influenzae type b to the nasopharynx, where the three-stranded Hib pilus filaments provide a robust tether to withstand coughs and sneezes . Cloning and Expression of the Gene for a Novel Protein from Mycobacterium smegmatis with Functional Similarity to Eukaryotic Calmodulin. Prasad T. Reddy, 2003.A calmodulin-like protein (CAMLP) from Mycobacterium smegmatis was purified to homogeneity and partially sequenced; these data were used to produce a full-length clone, whose DNA sequence contained a 55-amino-acid open reading frame . M . smegmatis CAMLP, expressed in Escherichia coli, exhibited properties characteristic of eukaryotic calmodulin: calcium-dependent stimulation of eukaryotic phosphodiesterase, which was inhibited by the calmodulin antagonist trifluoperazine, and reaction with anti-bovine brain calmodulin antibodies . Consistent with the presence of nine acidic amino acids (16%) in M . smegmatis CAMLP, there is one putative calcium-binding domain in this CAMLP, compared to four such domains for eukaryotic calmodulin, reflecting the smaller molecular size (approximately 6 kDa) of M . smegmatis CAMLP . Ultracentrifugation and mass spectral studies excluded the possibility that calcium promotes oligomerization of purified M . smegmatis CAMLP . Synergistic Effects of Cellulosomal Xylanase and Cellulases from Clostridium cellulovorans on Plant Cell Wall Degradation. Koichiro Murashima, 2003.Plant cell walls are comprised of cellulose and hemicellulose and other polymers that are intertwined, and this complex structure presents a barrier to degradation by pure cellulases or hemicellulases . In this study, we determined the synergistic effects on corn cell wall degradation by the action of cellulosomal xylanase XynA and cellulosomal cellulases from Clostridium cellulovorans . XynA minicellulosomes and cellulase minicellulosomes were found to degrade corn cell walls synergistically but not purified substrates such as xylan and crystalline cellulose . The mixture of XynA and cellulases at a molar ratio of 1:2 showed the highest synergistic effect of 1.6 on corn cell wall degradation . The amounts both of xylooligosaccharides and cellooligosaccharides liberated from corn cell walls were increased by the synergistic action of XynA and cellulases . Although synergistic effects on corn cell wall degradation were found in simultaneous reactions with XynA and cellulases, no synergistic effects were observed in sequential reactions . The possible mechanism of synergism between XynA and cellulases is discussed .
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