Killing of Mycolic Acid-Containing Bacteria Aborted Induction of Antibiotic Production by Streptomyces in Combined-Culture

Co-culture of Streptomyces with mycolic acid-containing bacteria (MACB), which we termed “combined-culture,” alters the secondary metabolism pattern in Streptomyces and has been a useful method for the discovery of bioactive natural products. In the course of our investigation to identify the inducing factor(s) of MACB, we previously observed that production of pigments in Streptomyces lividans was not induced by factors such as culture extracts or mycolic acids. Although dynamic changes occurred in culture conditions because of MACB, the activation of pigment production by S. lividans was observed in a limited area where both colonies were in direct contact. This suggested that direct attachment of cells is a requirement and that components on the MACB cell membrane may play an important role in the response by S. lividans. Here we examined whether this response was influenced by dead MACB that possess intact mycolic acids assembled on the outer cell membrane. Formaldehyde fixation and γ-irradiation were used to prepare dead cells that retain their shape and mycolic acids of three MACB species: Tsukamurella pulmonis, Rhodococcus erythropolis, and Rhodococcus opacus. Culturing tests verified that S. lividans does not respond to the intact dead cells of three MACB. Observation of combined-culture by scanning electron microscopy (SEM) indicated that adhesion of live MACB to S. lividans mycelia were a significant interaction that resulted in formation of co-aggregation. In contrast, in the SEM analysis, dead cells were not observed to adhere. Therefore, direct attachment by live MACB cells is proposed as one of the possible factors that causes Streptomyces to alter its specialized metabolism in combined-culture.

我们将含分枝菌酸细菌（mycolic acid-containing bacteria, MACB）与链霉菌的共培养体系命名为“联合培养”，该体系可改变链霉菌的次级代谢模式，是发掘生物活性天然产物的有效手段。在我们鉴定MACB诱导因子的研究过程中，此前曾观察到变铅青链霉菌（Streptomyces lividans）的色素产生无法通过培养物提取物或分枝菌酸等因子诱导。尽管MACB会使培养环境发生动态变化，但仅在两种菌落直接接触的有限区域内，才可观察到变铅青链霉菌的色素产生被激活。这提示细胞的直接附着是必要条件，MACB细胞膜上的组分可能在变铅青链霉菌的应答反应中发挥重要作用。本研究旨在探究该应答反应是否受保留了完整外膜组装分枝菌酸的死MACB影响。我们采用甲醛固定与γ射线辐照的方法，制备了3种MACB——肺土库曼菌（Tsukamurella pulmonis）、红平红球菌（Rhodococcus erythropolis）、迟钝红球菌（Rhodococcus opacus）——的形态完整且保留分枝菌酸的死细胞。培养实验证实，变铅青链霉菌不会对这3种MACB的完整死细胞产生应答。通过扫描电子显微镜（SEM）观察联合培养体系发现，活MACB与变铅青链霉菌菌丝体的黏附是显著的相互作用，可形成共聚集结构。与之相反，扫描电子显微镜分析未观察到死细胞发生黏附。因此，我们提出活MACB细胞的直接附着，是引发链霉菌在联合培养中改变其特化代谢的潜在因素之一。