Abstract

Two-dimensional gel electrophoresis was used to follow changes in gene expression associated with antibiotic (bialaphos) biosynthesis in Streptomyces hygroscopicus. Cultures were pulse-labelled with [35S]-methionine before, during, and after the switch from primary to secondary metabolism in order to compare kinetic profiles of bialaphos (antibiotic) production (bap) genes during this metabolic transition. Separation of gene products on two-dimensional gels revealed that 27 were dependent on brpA for optimal expression and were activated as the culture approached stationary phase. Genes which encoded 10 brpA-dependent proteins were mapped to a 10 kb SstI fragment of the 35 kb bap gene cluster by expressing them in Streptomyces lividans using the thiostrepton-inducible tipA promoter. N-terminal amino acid sequences of two brpA-dependent proteins, obtained by direct microsequencing of protein spots excised from two-dimensional gels, identified them as gene products mapping to the same region and involved in secondary metabolic conversions of the bap pathway. The kinetics of synthesis of 16 brpA-dependent gene products were characterized using QUEST computer software. Cluster analysis performed on the kinetics of synthesis of 346 of the most highly expressed gene products of HP5-29, including 16 brpA-dependent ones, identified 75 families having distinct patterns of expression. Many brpA-dependent proteins were clustered together; 10 were found in one kinetic family. These kinetic families also included brpA-independent gene products perhaps subject to similar regulatory mechanisms and thus possibly involved in bialaphos biosynthesis. The activation/derepression of bap expression took place as cultures approached stationary phase and was temporally related to synthesis of ppGpp.