Mechanism-related changes in the gene transcription and protein synthesis patterns of Haemophilus influenzae after treatment with transcriptional andtranslational inhibitors

High-resolution two-dimensional gel electrophoresis of pulse-labeled Haemop hilus influenzae extracts allows for the separation and quantification of m ore than five hundred protein spots. We have determined the changes in the protein synthesis patterns triggered by treatment with inhibitors of transc ription, Rifampicin (Rif) and translation, Chloramphenicol (Chl), Erythromy cin (Ery), Fusidate (Fus), Puromycin (Pur), Kanamycin (Kan), Streptomycin ( Str), and Tetracycline (Tet) relative to the total protein synthesis rate. More than 200 spots changed in intensity under at least one condition. With the exception of the aminoglycosides, Kan and Str, all inhibitors triggere d a clear increase in the synthesis rates of ribosomal proteins and RNA pol ymerase subunits. Northern analysis of rpoA, rpoB, rpoC, and six ribosomal protein genes indicated induction of transcription as well as antiterminati on as part of the mechanism of the regulation of gene expression. Total RNA synthesis was increased after exposure to Chi, fry, Fus, and Tet, whereas Str had no effect. Rif led to an almost complete shutdown of RNA synthesis. Exposure to Chi, fry, Fus, Rif, and Tet resulted in a decrease in the conc entration of the stringent factor, guanosine 5',3'-bis-diphosphate (ppGpp) whereas Str again had no effect. Thus, as in Escherichia coli, the response of H. influenzae to translational inhibitors appears to be mediated by the regulatory nucleotide ppGpp.