Simultaneous coexpression of Borrelia burgdorferi Erp proteins occurs through a specific, erp locus-directed regulatory mechanism.
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An individual Borrelia burgdorferi bacterium can encode as many as 13 different Erp (OspE/F-related) proteins from mono-and bicistronic loci that are carried on up to 10 separate plasmids. We demonstrate through multilabel immunofluorescence analyses that individual bacteria simultaneously coexpress their entire Erp protein repertoire. While it has been proposed that B. burgdorferi controls expression of Erp and other plasmid-encoded proteins through changes in DNA topology, we observed regulated Erp expression in the absence of detectable differences in DNA supercoiling. Likewise, inhibition of DNA gyrase had no detectable effect on Erp expression. Furthermore, expression of loci physically adjacent to erp loci was observed to be independently regulated. It is concluded that Erp expression is regulated by a mechanism(s) directed at erp loci and not by a global, plasmid-wide mechanism. (+info)
Methyltransferase genes in Streptomyces rishiriensis: new coumermycin derivatives from gene-inactivation experiments.
(10/101)
The coumarin antibiotic coumermycin A(1) contains at least eight methyl groups, presumably derived from S-adenosylmethionine. Two putative methyltransferase genes, couO and couP, of the coumermycin A(1) biosynthetic gene cluster were inactivated by in-frame deletion. In the resulting mutants, coumermycin A(1) production was abolished. New coumermycin derivatives were accumulated instead, and were identified by HPLC-MS using selected reaction monitoring via electrospray ionization. couO mutants accumulated a coumermycin derivative lacking the methyl groups at C-8 of the characteristic aminocoumarin rings, whereas in the couP mutant a coumermycin derivative lacking the methyl groups at the 4-hydroxyl groups of the two deoxysugar moieties was identified. These results provided evidence that couO encodes a C-methyltransferase responsible for the transfer of a methyl group to C-8 of the aminocoumarin ring, and couP an O-methyltransferase for methylation of 4-OH of the sugar in the biosynthesis of coumermycin A(1), respectively. C-methylation of the aminocoumarin ring is considered as an early step of coumermycin biosynthesis. Nevertheless, the intermediates with the non-methylated aminocoumarin ring were accepted by the enzymes catalysing the subsequent steps of the pathway. The new, demethylated secondary metabolites were produced in an amount at least as high as that of coumermycin A(1) in the wild-type. (+info)
groEL expression in gyrB mutants of Borrelia burgdorferi.
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GroEL protein and groEL mRNA transcript were up-regulated in gyrB mutants of Borrelia burgdorferi, a causative agent of Lyme disease. Furthermore, the protein and transcript levels in gyrB mutants were greater than those in experimentally heat-shocked cultures of wild-type B. burgdorferi. Circular DNA in the gyrB mutants was more relaxed than in wild-type cells, although groEL is on the linear chromosome of B. burgdorferi. To our knowledge, this is the first evidence, albeit indirect, for the effect of DNA topology on gene expression from a linear DNA molecule in a bacterium. (+info)
Alternative splicing controls the mechanisms of FAK autophosphorylation.
(12/101)
Focal adhesion kinase (FAK) is activated following integrin engagement or stimulation of transmembrane receptors. Autophosphorylation of FAK on Tyr-397 is a critical event, allowing binding of Src family kinases and activation of signal transduction pathways. Tissue-specific alternative splicing generates several isoforms of FAK with different autophosphorylation rates. Despite its importance, the mechanisms of FAK autophosphorylation and the basis for differences between isoforms are not known. We addressed these questions using isoforms of FAK expressed in brain. Autophosphorylation of FAK(+), which is identical to that of "standard" FAK, was intermolecular in transfected cells, although it did not involve the formation of stable multimeric complexes. Coumermycin-induced dimerization of gyrase B-FAK(+) chimeras triggered autophosphorylation of Tyr-397. This was independent of cell adhesion but required the C terminus of the protein. In contrast, the elevated autophosphorylation of FAK(+6,7), the major neuronal splice isoform, was not accounted for by transphosphorylation. Specifically designed immune precipitate kinase assays confirmed that autophosphorylation of FAK(+) was intermolecular, whereas autophosphorylation of FAK(+6,7) or FAK(+7) was predominantly intramolecular and insensitive to the inhibitory effects of the N-terminal domain. Our results clarify the mechanisms of FAK activation and show how alternative splicing can dramatically alter the mechanism of autophosphorylation of a protein kinase. (+info)
Molecular cloning and sequence analysis of the clorobiocin biosynthetic gene cluster: new insights into the biosynthesis of aminocoumarin antibiotics.
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The biosynthetic gene cluster of the aminocoumarin antibiotic clorobiocin was cloned by screening of a cosmid library of Streptomyces roseochromogenes DS 12.976 with two heterologous probes from the novobiocin biosynthetic gene cluster. Sequence analysis revealed 27 ORFs with striking similarity to the biosynthetic gene clusters of novobiocin and coumermycin A(1). Inactivation of a putative aldolase gene, cloR, by in-frame deletion led to the abolishment of the production of clorobiocin. Feeding of the mutant with 3-dimethylallyl-4-hydroxybenzoic acid (Ring A of clorobiocin) restored clorobiocin production. Here, it is suggested that the formation of Ring A of clorobiocin may proceed via a retro-aldol reaction catalysed by CloR, i.e. by a mechanism different from the previously elucidated benzoic acid biosynthetic pathway in Streptomyces maritimus. A comparison of the gene clusters for clorobiocin, novobiocin and coumermycin A(1) showed that the structural differences between the three antibiotics were reflected remarkably well by differences in the organization of their respective biosynthetic gene clusters. (+info)
Resistance genes of aminocoumarin producers: two type II topoisomerase genes confer resistance against coumermycin A1 and clorobiocin.
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The aminocoumarin resistance genes of the biosynthetic gene clusters of novobiocin, coumermycin A(1), and clorobiocin were investigated. All three clusters contained a gyrB(R) resistance gene, coding for a gyrase B subunit. Unexpectedly, the clorobiocin and the coumermycin A(1) clusters were found to contain an additional, similar gene, named parY(R). Its predicted gene product showed sequence similarity with the B subunit of type II topoisomerases. Expression of gyrB(R) and likewise of parY(R) in Streptomyces lividans TK24 resulted in resistance against novobiocin and coumermycin A(1), suggesting that both gene products are able to function as aminocoumarin-resistant B subunits of gyrase. Southern hybridization experiments showed that the genome of all three antibiotic producers and of Streptomyces coelicolor contained two additional genes which hybridized with either gyrB(R) or parY(R) and which may code for aminocoumarin-sensitive GyrB and ParY proteins. Two putative transporter genes, novA and couR5, were found in the novobiocin and the coumermycin A(1) cluster, respectively. Expression of these genes in S. lividans TK24 resulted in moderate levels of resistance against novobiocin and coumermycin A(1), suggesting that these genes may be involved in antibiotic transport. (+info)
Agonist-independent desensitization and internalization of the human platelet-activating factor receptor by coumermycin-gyrase B-induced dimerization.
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Platelet-activating factor (PAF) is a phospholipid with potent and diverse physiological actions, particularly as a mediator of inflammation. We have reported previously that mutant G protein-coupled receptors (GPCRs) affect the functional properties of coexpressed wild-type human PAF receptor (hPAFR) (Le Gouill, C., Parent, J. L., Caron, C. A., Gaudreau, R., Volkov, L., Rola-Pleszczynski, M., and Stankova, J. (1999) J. Biol. Chem. 274, 12548-12554). Increasing evidence suggests that dimerization of GPCRs may play an important role in the regulation of their biological activity. Additional data have also suggested that dimerization may be important in the subsequent internalization of the delta-opioid receptor. To investigate the specific role of dimerization in the internalization process of GPCRs, we generated a fusion protein of hPAFR and bacterial DNA gyrase B (GyrB), dimerized through the addition of coumermycin. We found that dimerization potentiates PAF-induced internalization of hPAFR-GyrB in Chinese hamster ovary cells stably expressing c-Myc-hPAFR-GyrB. Coumermycin-driven dimerization was also sufficient to induce an agonist-independent sequestration process in an arrestin- and clathrin-independent manner. Moreover, the protein kinase C inhibitors staurosporine and GF109203X blocked the coumermycin-induced desensitization of hPAFR-GyrB, suggesting the implication of protein kinase C in the molecular mechanism mediating the agonist-independent desensitization of the receptor. Taken together, these findings suggest a novel mechanism of GPCR desensitization and internalization triggered by dimerization. (+info)
CloR, a bifunctional non-heme iron oxygenase involved in clorobiocin biosynthesis.
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The aminocoumarin antibiotics novobiocin and clorobiocin contain a 3-dimethylallyl-4-hydroxybenzoate (3DMA-4HB) moiety. The biosynthesis of this moiety has now been identified by biochemical and molecular biological studies. CloQ from the clorobiocin biosynthetic gene cluster in Streptomyces roseochromogenes DS 12976 has recently been identified as a 4-hydroxyphenylpyruvate-3-dimethylallyltransferase. In the present study, the enzyme CloR was overexpressed in Escherichia coli, purified, and identified as a bifunctional non-heme iron oxygenase, which converts 3-dimethylallyl-4-hydroxyphenylpyruvate (3DMA-4HPP) via 3-dimethylallyl-4-hydroxymandelic acid (3DMA-4HMA) to 3DMA-4HB by two consecutive oxidative decarboxylation steps. In 18O2 labeling experiments we showed that two oxygen atoms are incorporated into the intermediate 3DMA-4HMA in the first reaction step, but only one further oxygen is incorporated into the final product 3DMA-4HB during the second reaction step. CloR does not show sequence similarity to known oxygenases. It apparently presents a novel member of the diverse family of the non-heme iron (II) and alpha-ketoacid-dependent oxygenases, with 3DMA-4HPP functioning both as an alpha-keto acid and as a hydroxylation substrate. The reaction catalyzed by CloR represents a new pathway for the formation of benzoic acids in nature. (+info)