The mupirocin trans-AT polyketide synthase pathway, provides a model system for manipulation of antibiotic biosynthesis. Its final phase involves removal of the tertiary hydroxyl group from pseudomonic acid B, PA-B, producing the fully active PA-A in a complex series of steps. To further clarify requirements for this conversion, we fed extracts containing PA-B to mutants of the producer strain singly deficient in each mup gene. This additionally identified mupM and mupN as required plus the sequence but not enzymic activity of mupL and ruled out need for other mup genes. A plasmid expressing mupLMNOPVCFU + macpE together with a derivative of the producer P. fluorescens strain NCIMB10586 lacking the mup cluster allowed conversion of PA-B to PA-A. MupN converts apo-mAcpE to holo-form while MupM is a mupirocin-resistant isoleucyl tRNA synthase, preventing self-poisoning. Surprisingly, the expression plasmid failed to allow the closely related P. fluorescens strain SBW25 to convert PA-B to PA-A.
Aims: To evaluate the efficiency of hel gene polymerase chain reaction (PCR) to detect Haemophilus influenzae in various clinical/non-clinical samples. Methods and Results: Seventy-four clinical samples (cerebrospinal fluid, blood, sputum, throat and nasal swabs) and throat swabs of 17 asymptomatic carriers were collected. Primers were used to amplify the hel gene of H. influenzae encoding P4 outer membrane protein directly from the processed samples. The samples were also examined by conventional culture methods and the results were compared with those of PCR. The culture methods showed positive results in 60 (65AE9%) of 91 samples in contrast to 62 (68AE12%) samples tested positive by PCR. None of the culture-positive samples were PCR-negative while two of the culture-negative samples were PCRpositive. The specificity of the products was confirmed by Southern hybridization and failure of various other organisms to amplify the hel gene product. The sensitivity of the PCR assay was found to be 50 pg of DNA. Conclusions: These findings suggest that the hel gene PCR is a rapid, sensitive and a specific new method for direct identification of H. influenzae. Significance and Impact of the study: Thus, this PCR test can improve the detection rate of H. influenzae in suspected clinical samples as compared with that of conventional culture methods.
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