Growth-Regulated Expression of a Bacteriocin,Produced by the Sweet Potato Pathogen Streptomyces ipomoeae,That Exhibits Interstrain Inhibition

Certain strains of the bacterial sweet potato pathogen Streptomyces ipomoeae produce the bacteriocin ipomicin, which inhibits other sensitive strains of the same species. Within the signal-sequence-encoding portion of the ipomicin structural gene ipoA exists a single rare TTA codon, which is recognized in Streptomyces bacteria by the temporally accumulating bldA leucyl tRNA. In this study, ipomicin was shown to stably accumulate in culture supernatants of S. ipomoeae in a growth-regulated manner that did not coincide with the pattern of ipoA expression. Similar growth-regulated production of ipomicin in Streptomyces coelicolor containing the cloned ipoA gene was found to be directly dependent on translation of the ipoA TTA codon by the bldA leucyl tRNA. The results here suggest that bldA-dependent translation of the S. ipomoeae ipoA gene leads to growth-regulated production of the ipomicin precursor, which upon processing to the mature form and secretion stably accumulates in the extracellular environment. To our knowledge, this is the first example of bldA regulation of a bacteriocin in the streptomycetes.Streptomyces bacteria are gram-positive spore-forming soil bacteria which display a complex life cycle (4, 6). Spore germination leads to vegetative growth as tangled masses of substrate mycelia on solid surfaces. Nutrient limitation triggers production of secondary metabolites as well as growth of vertically extending aerial hyphae, which are morphologically distinct filaments that derive nutrients from the dying substrate cell layer. Eventually, individual aerial hyphae differentiate into chains of spores. Streptomyces mycelia growing in liquid media display a multiphasic growth curve similar to that of unicellular bacteria. As the culture enters stationary phase, Streptomyces bacteria produce secondary metabolites but do not typically differentiate morphologically (2).Streptomyces ipomoeae is the causative agent of soil rot, the widespread and destructive disease of sweet potatoes. The disease results in decay of fibrous feeder roots and development of necrotic lesions on the edible storage roots (9). Prevention of soil rot currently relies solely on development of resistant sweet potato cultivars; however, alternative approaches, including the use of biocontrol methods, are now beginning to be investigated.Previously, 36 strains of S. ipomoeae were divided into three groups based on interstrain inhibition during their cocultivation on agar plates (7). The group III strains were found to produce a diffusible 10-kDa bacteriocin-like protein (ipomicin), which is inhibitory to group I and II members and which is stable in culture supernatants for at least 1 h at 40°C (in a pH range of 6 to 10) (26). Sequence analyses of the ipomicin structural gene (ipoA) and its protein product revealed that ipomicin is initially expressed in precursor form, which upon processing of an N-terminal signal sequence becomes the 10-kDa mature form (26).Given its potential as a biocontrol agent, we were interested in elucidating the expression characteristics of ipomicin. It was noted previously that TTA, the rarest codon found within the GC-rich Streptomyces genus, is present once in ipoA within the region that designates the ipomicin signal sequence (26). TTA codons in Streptomyces bacteria are recognized by a single leucyl tRNA species encoded by the gene bldA, which is required for normal morphological and physiological differentiation in these organisms (5, 14). The latter effect is due in part to the fact that regulatory genes of antibiotic clusters and aerial hyphae development also contain TTA codons (5). In the model streptomycete, Streptomyces coelicolor, bldA has been shown to be an effective growth phase regulator because it is expressed initially in liquid and surface cultures as an inactive precursor and, upon processing, begins to temporally accumulate as a mature tRNA, at least under certain physiological conditions (15, 24). Interestingly, TTA-containing genes show variation in their dependence on bldA for expression, with some genes demonstrating strong dependence while others show only partial or no apparent dependence (14, 23).Here, stable ipomicin protein is shown to be temporally produced during S. ipomoeae growth in a manner inconsistent with ipoA mRNA levels. Similar contrary results for ipomicin protein and ipoA mRNA concentrations were observed during growth of the heterologous host S. coelicolor expressing a cloned version of the S. ipomoeae ipoA gene, and this effect was shown to be directly dependent on translation of the ipoA TTA codon by the bldA leucyl tRNA. The data here suggest that regulation by bldA leads to temporal production of the ipomicin precursor, which when processed to the mature form is secreted to the external environment, where it accumulates.