Nucleotide sequence of the rci gene encoding shufflon-specific DNA recombinase in the IncI1 plasmid R64: homology to the site-specific recombinases of integrase family

Summary Shufflon is a novel type of DNA rearrangement in which four DNA segments are flanked by seven 19-bp repeat sequences. The site-specific recombination between any inverted repeats results in an inversion of the DNA segment(s) either independently or in groups. The recombination is mediated by a gene designated rci. We have determined the nucleotide sequence of the rci gene and found that it encodes a basic protein with 384 amino acid residues. The rci gene was fused with lacZ and its gene product was identified by Western blot analysis. The Rci protein shows regional homologies to the site-specific recombinases encoded by the bacteriophage genomes, including those of , 80, P22, P2, 186, P4 and P1.     

Analysis of the <Emphasis Type="Italic">pilU</Emphasis> gene for the prepilin peptidase involved in the biogenesis of type IV pili encoded by plasmid R64

In many type IV pili, the N-terminal amino acid of the pilin subunit is N-methylated phenylalanine. A prepilin peptidase removes the leader peptide from the precursor and methylates the amino group of the newly formed phenylalanine. PilS, the precursor of the pilin encoded by plasmid R64, is processed by the prepilin peptidase PilU, but the N-terminal amino acid of the mature pilin is a non-methylated tryptophan that is otherwise modified. To study the relationship between the structure and function of PilU, 42 missense pilU mutations were constructed by PCR and site-directed mutagenesis, and the ability of these pilU mutants to complement a pilU null mutant for mating in liquid culture was analyzed. Although practically no conjugation was noted for 21 of the mutants, the remaining 21 supported varying levels of residual plasmid transfer activity. Two mutants with aspartic acid replacements in conserved motifs exhibited no PilU activity, suggesting that the product of the pilU gene is an aspartic acid peptidase, like TcpJ, the prepilin peptidare of Vibrio cholerae. No PilS processing was detected in 21 of the mutants, but the remaining 21 exhibited varying levels of residual PilS processing. A close correlation was noted between residual PilS processing activity and conjugative transfer, suggesting that the pilU gene product possesses prepilin peptidase activity, but is unable to methylate the N-terminal tryptophan. Based on the activity of pilU-phoA and pilU-lacZ fusion genes encoding different segments of PilU, a model for the membrane topology of the protein is also proposed. Furthermore, some amino acid substitutions in the pilU portion of the pilU-phoA and pilU-lacZ fusion genes were found to alter the membrane topology of the product.     

Identification of components of a new stability system of plasmid R1, ParD, that is close to the origin of replication of this plasmid

Summary We provide evidence that a mutation which derepresses an autoregulated system that is located in the vicinity of the basic replicon of R1, stabilizes the ParA^- and ParB^- miniplasmid of R1 pKN1562, without increasing its copy number. The system, which we have called ParD, maps inside the 1.45-kb PstI-EcoRI fragment that is adjacent to the origin of replication of the plasmid. Two protiens whose expression is coordinated are components of the system. The sequence of the PstI-EcoRI fragment was obtained. The wild-type ParD system determines in cis a basal but detectable stability.     

Development of a high-performance liquid chromatographic–mass spectrometric assay for the specific and sensitive quantification of Ro 64-0802, an anti-influenza drug, and its pro-drug, oseltamivir, in human and animal plasma and urine

Oseltamivir phosphate (Ro 64-0796/002) is a pro-drug of the anti-influenza neuraminidase inhibitor, Ro 64-0802, and as Tamiflu™, has been developed for the treatment of both A and B strains of the disease. This paper describes an HPLC–MS–MS assay for both compounds in plasma and urine which fulfils all of the criteria for a good analytical method. It is sensitive with limits of quantification of 1 and 10 ng/ml for the pro-drug and active neuraminidase inhibitor, respectively. It is both accurate and precise with typical coefficients of variation from some 5000 quality control samples of approximately ±3 and ±6%, respectively. Extensive stability studies have demonstrated the absence of significant problems associated with the decomposition of either compound, although ex vivo hydrolysis of Ro 64-0796 to Ro 64-0802 in rodent plasma has to be prevented by the use of the esterase inhibitor, dichlorvos.     

The response to a specific germinant by <Emphasis Type="Italic">Bacillus anthracis</Emphasis> spores in primary mouse macrophages is modulated by a protein encoded on the pXO1 plasmid

A Bacillus anthracis Sterne pXO1 plasmid-encoded protein designated Cot43 was found in coat extracts of purified spores. Cot43 is a tetratricopeptide repeat domain protein related to those which function as phosphatases in the sporulation phosphorelay and as regulators of competence and pathogenic factors. The synthesis of Cot43 began in the late exponential phase downstream from a sigmaA promoter (as mapped by RACE) and it was present at least until the formation of phase white endospores. There was specificity in the association of Cot43 with B. anthracis spores since Bacillus cereus producing Cot43 from a cloned gene had very little of this protein in spore coat extracts. In addition, Cot43 was synthesized by B. anthracis cells to the same extent in glucose-yeast extract and nutrient sporulation media, but was essentially absent from spores formed in the former. l-histidine is an important germinant for B. anthracis spores in macrophages, Spores produced by a mutant with a disruption of cot43 germinated in response to l-histidine both in vitro and within primary mouse macrophages earlier and more extensively than Sterne strain spores. The germination delay due to the presence of Cot43 would enhance spore survival and thus increase the chances for a successful infection. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The Mr 17500 region of the A chain of urokinase is required for interaction with a specific receptor in A431 cells

We have found the existence of specific receptors for the plasminogen activator, urokinase, in A431 human epidermoid carcinoma cells, cultures in plasminogen-free conditions. Two subsets of receptors have been recognized on the basis of 125I-labelled urokinase binding analysis: about 1 X 10(3) high-affinity (Kd = 5.0 X 10(-11) M) and 1 X 10(5) low-affinity (Kd = 9 X 10(-9) M) receptors per cell. The electron microscopic observation of a urokinase: ferritin conjugate has shown single and clustered receptors at the cell surface. Down-regulation of the receptors (T1/2 = 3.77 h) follows the binding of urokinase. The binding does not involve an intact catalytic site and is inhibited by a monoclonal antibody against the Mr 17500 proteolytic fragment of the A chain of urokinase.