Germination and Growth Analysis of Streptomyces lividans at the Single-Cell Level Under Varying Medium Compositions

Abstract

Quantitative single-cell cultivation has provided fundamental contributions to our understanding of heterogeneity among industrially used microorganisms. Filamentous growing Streptomyces species are emerging platform organisms for industrial production processes, but their exploitation is still limited due to often reported high batch-to-batch variations and unexpected growth and production differences. Population heterogeneity is suspected to be one responsible factor, which is so far not systematically investigated at the single-cell level. Novel microfluidic single-cell cultivation devices offer promising solutions to investigate these phenomena. In this study, we investigated the germination and growth behavior of Streptomyces lividans TK24 under varying medium compositions on different complexity levels (i.e., mycelial growth, hyphal growth and tip elongation) on single-cell level. Our analysis reveals a remarkable stability within growth and germination of spores and early mycelium development when exposed to constant and defined environments. We show that spores undergo long metabolic adaptation processes of up to > 30 h to adjust to new medium conditions, rather than using a “persister” strategy as a possibility to cope with rapidly changing environments. Due to this uniform behavior, we conclude that S. lividans can be cultivated quite robustly under constant environmental conditions as provided by microfluidic cultivation approaches. Failure and non-reproducible cultivations are thus most likely to be found in less controllable larger-scale cultivation workflows and as a result of environmental gradients within large-scale cultivations.

mycelyso – high-throughput analysis of Streptomyces mycelium live cell imaging data

“…mycelyso has been used to analyze a large-scale screening study to quantify the impact of six different growth media compositions on the hyphae-level growth behavior of Streptomyces lividans TK24 [16]. For the description of the imaging conditions and the biotechnological findings derived from the data, the reader is referred to the study [16]. Within this software publication, we focus on presenting performance measures for the mycelyso software.…”

“…These individual hyphae-level tracks further provide intra-mycelium growth parameters, such as a tip growth rates of 16.33±6.91 µ m·h −1 . In comparison, the overall growth rate taking multiple on-chip positions (n=10) into account was determined to be 0.38±0.11 h −1 [16], and the germination delay 10.0±2.4 h.…”

“…Hyphae-level metrics unravel the precise kinetics of growth and response of the organism to the selected cultivation conditions, and provide insights into the formation of macroscopic phenotypes. In the larger study [16], from which the example dataset was taken, six different media compositions (one complex medium and five different minimal media) were imaged over time frames of 24–48 h. This setup yielded 675 image stacks in total. The image stacks were processed with mycelyso as described before and generated the biotechnological key parameters needed for the study in an automated manner.…”

“…Diverse growth metrics can be extracted, with minimal manual burden and maximal reproducibility. The utility of mycelyso has been proven in a comprehensive screening study with Streptomyces lividans TK24 [16] where we analyzed the influence of different growth media compositions on the growth behavior from whole mycelium to individual hyphae-level.…”

Characterization of Sigma Factor Genes in Streptomyces lividans TK24 Using a Genomic Library-Based Approach for Multiple Gene Deletions

“…S. lividans TK24 was derived from S. lividans strain 66 after UV mutagenesis and protoplast regeneration and, like its parental strain, is producing actinorhodin, the calcium-dependent antibiotic and undecylprodigiosin only under certain growth conditions and with a low titer (Hopwood et al, 1983). Due to low endogenous protease activity and reproducible growth behavior during cultivation in constant environmental conditions, S. lividans TK24 is an excellent host strain for heterologous secretion of proteins (Anné et al, 2017; Koepff et al, 2018). Despite being used for decades as a host for secondary metabolites and protein production, in basic research S. coelicolor was preferred over S. lividans strains.…”