Methylenomycin production by Streptomyces coelicolor A3(2) may be triggered by either of two environmental signals: alanine growth-rate-limiting conditions and/or an acidic pH shock. The production of this SCP1-encoded antibiotic was studied by using batch and chemostat cultures. Batch cultures indicated a role for both nutritional status and culture pH in its regulation. Steady-state methylenomycin production and transcription of an mmy gene under alanine but not glucose growth-rate-limiting conditions was demonstrated in chemostat culture. Transient mmy expression and methylenomycin production occurred following an acidic pH shock. This stimulation of methylenomycin production occurred independently of the nutritional status of the growth environment. Antibiotic production was partially suppressed under alanine compared with glucose growthrate-limiting conditions following the acidic pH shock. A low specific growth rate was a prerequisite for both steady-state and transient production of methylenomycin.The traditional view of antibiotic production in Streptomyces bacteria is that it is restricted to the stationary phase (idiophase) of the batch growth cycle and is triggered by environmental factors which give rise to this state (reviewed in reference 2). This view has arisen from the extensive use of batch culture in academic studies and industrial processes. To decipher the mechanisms whereby Streptomyces bacteria sense changes in their environment and transduce them into expression of specific secondary metabolic gene sets, it is essential to clearly define the environmental conditions which trigger antibiotic production. The dynamic behavior of batch cultures (23) hinders this effort.A number of authors (1,8,10,16,18,21,24,28,31,32) have studied antibiotic production by streptomycetes by using chemostat culture. From these studies, it is clear that secondary metabolism is not strictly associated with zero growth (22) and that antibiotic production is subject to a growth rate dependence. Also, in a number of cases, antibiotic production has been demonstrated to be influenced by the nutritional status of the growth environment and hence is dependent on a type of catabolite repression or metabolite interference (2). However, in many cases, researchers have reported the presence of metabolite interference based solely on studies using batch culture.The antibiotic methylenomycin is one of at least five secondary metabolites synthesized by Streptomyces coelicolor A3(2) (33). Its biosynthetic genes (mmy), together with a resistance determinant (mmr), are all carried on a large, linear plasmid, SCP1 (17). In a previous report (12), we described a set of nutritional conditions which permitted the biosynthesis of methylenomycin in batch culture. Methylenomycin was produced toward the end of the active growth phase but prior to the complete consumption of the growth stoichiometrically limiting substrate, alanine. In these experiments, the pH was not controlled and decreased during the growth phase from 7.2 to 5.5. Low...