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Evidence for Lateral Transfer of the Suilysin Gene Region of Streptococcus suis. Daisuke Takamatsu, 2002.Suilysin is a cholesterol-binding cytolysin encoded by sly in Streptococcus suis . DNA sequence determination of the sly locus in a strain lacking sly revealed the presence of another gene, designated orf102, in the place of sly . No transposable element or long-repeat sequence was found in the close vicinity . Except for six strains whose corresponding loci have been rearranged, all of the remaining 62 strains examined had either sly or orf102 at the same locus and their flanking regions were conserved . The genetic organizations having either sly or orf102 were found in the strains whose 16S rRNA sequences were identical . These results suggest that S . suis acquired sly or orf102 from a foreign source and that these genes subsequently spread among S . suis strains by homologous recombination . Genus-Specific Protein Binding to the Large Clusters of DNA Repeats (Short Regularly Spaced Repeats) Present in Sulfolobus Genomes. Xu Peng, 2003.Short regularly spaced repeats (SRSRs) occur in multiple large clusters in archaeal chromosomes and as smaller clusters in some archaeal conjugative plasmids and bacterial chromosomes . The sequence, size, and spacing of the repeats are generally constant within a cluster but vary between clusters . For the crenarchaeon Sulfolobus solfataricus P2, the repeats in the genome fall mainly into two closely related sequence families that are arranged in seven clusters containing a total of 441 repeats which constitute ca . 1% of the genome . The Sulfolobus conjugative plasmid pNOB8 contains a small cluster of six repeats that are identical in sequence to one of the repeat variants in the S . solfataricus chromosome . Repeats from the pNOB8 cluster were amplified and tested for protein binding with cell extracts from S . solfataricus . A 17.5-kDa SRSR-binding protein was purified from the cell extracts and sequenced . The protein is N terminally modified and corresponds to SSO454, an open reading frame of previously unassigned function . It binds specifically to DNA fragments carrying double and single repeat sequences, binding on one side of the repeat structure, and producing an opening of the opposite side of the DNA structure . It also recognizes both main families of repeat sequences in S . solfataricus . The recombinant protein, expressed in Escherichia coli, showed the same binding properties to the SRSR repeat as the native one . The SSO454 protein exhibits a tripartite internal repeat structure which yields a good sequence match with a helix-turn-helix DNA-binding motif . Although this putative motif is shared by other archaeal proteins, orthologs of SSO454 were only detected in species within the Sulfolobus genus and in the closely related Acidianus genus . We infer that the genus-specific protein induces an opening of the structure at the center of each DNA repeat and thereby produces a binding site for another protein, possibly a more conserved one, in a process that may be essential for higher-order stucturing of the SRSR clusters . A Cytochrome c from a Lupanine-Transforming Pseudomonas putida Strain Is Expressed in Escherichia coli during Aerobic Cultivation and Efficiently Exported and Assembled in the Periplasm. Mustak A. Kaderbhai, 2003.We have cloned, sequenced, and heterologously expressed a periplasmic cytochrome c from a lupanine-utilizing Pseudomonas putida strain . Aerobic batch cultivation of Escherichia coli TB1 harboring the cytochrome c gene placed downstream of the lac promoter in pUC9 vector resulted in significant production of the holo-cytochrome c in the periplasm (  4 mg of hemoprotein/liter of culture) . The recombinant cytochrome c was purified to homogeneity and was found to be functional in accepting electrons from lupanine hydroxylase while catalyzing hydroxylation of lupanine . Comparison of the N-terminal amino acid sequence of the isolated cytochrome c with that deduced from the DNA sequence indicated that the signal sequence was processed at the bond position predicted by the SigPep program . The molecular size of the cytochrome c determined by electrospray mass spectrometry (9,595) was in precise agreement with that predicted from the nucleotide sequence .
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