Physiological and cell morphology adaptation of Bacillus subtilis at near‐zero specific growth rates: a transcriptome analysis

Abstract

Nutrient scarcity is a common condition in nature, but the resulting extremely low growth rates (below 0.025 h(-1) ) are an unexplored research area in Bacillus subtilis. To understand microbial life in natural environments, studying the adaptation of B. subtilis to near-zero growth conditions is relevant. To this end, a chemostat modified for culturing an asporogenous B. subtilis sigF mutant strain at extremely low growth rates (also named a retentostat) was set up, and biomass accumulation, culture viability, metabolite production and cell morphology were analysed. During retentostat culturing, the specific growth rate decreased to a minimum of 0.00006 h(-1) , corresponding to a doubling time of 470 days. The energy distribution between growth and maintenance-related processes showed that a state of near-zero growth was reached. Remarkably, a filamentous cell morphology emerged, suggesting that cell separation is impaired under near-zero growth conditions. To evaluate the corresponding molecular adaptations to extremely low specific growth, transcriptome changes were analysed. These revealed that cellular responses to near-zero growth conditions share several similarities with those of cells during the stationary phase of batch growth. However, fundamental differences between these two non-growing states are apparent by their high viability and absence of stationary phase mutagenesis under near-zero growth conditions.

Genome-Wide Transcriptional Responses to Carbon Starvation in Nongrowing Lactococcus lactis

“…This response can be classified as a typical stringent response (17), which was not elicited under near-zero-growth conditions but was apparent only upon subsequent starvation. These observations are in clear contrast to the stringent response that is already significantly induced during retentostat cultivation of another Gram-positive bacterium, Bacillus subtilis (39), but also in cultures of Saccharomyces cerevisiae (40). Despite the starvation induced stringent response, the L. lactis culture viability remained relatively high, and the culture was capable of rapid recovery of metabolic activity upon reinitiation of the medium supply, which also led to a relief of the stringent response.…”

Physiological and Transcriptional Responses of Different Industrial Microbes at Near-Zero Specific Growth Rates

“…Similarly, the morphology of B. subtilis cells changed from the characteristic short rod shape to substantially elongated cells after 42 days of retentostat cultivation ( Fig. 4E and F) (40). Conversely, microscopic examination of chemostat-and retentostat-grown L. plantarum cells did not reveal significant changes in morphology ( Fig.…”

“…2 (5,6). Remarkably, the viability of B. subtilis remained above 90% as well, while the fraction of cultivable cells decreased to 30% (40). This is most probably due to the observed cell-chain formation under these conditions, which strongly confounds accurate CFU enumeration, rather than to entry of these cells into a viable but nonculturable state (41).…”

“…The major catabolic products were lactate and low concentrations of acetate, formate, and ethanol for L. plantarum (6), CO 2 and H 2 O for B. subtilis (40), and ethanol and CO 2 for S. cerevisiae (29). In contrast, substantial fluctuations in fermentation products were observed in L. lactis cultures that were also reflected by modulations in the expression of the corresponding genes.…”

“…5). Both in the eukaryotic and prokaryotic kingdoms, dedicated glucose-sensing and signaling cascades cultivation, prolonged glucose limitation led to the progressive relief of repression of CcpA targets in prokaryotes and of glucoserepressible genes in S. cerevisiae, thereby demonstrating a gradual alleviation of carbon catabolite repression when approaching (near-)zero growth (4,6,40,53). In L. lactis, this carbon catabolite repression transcriptional relief was shown to enable retentostatderived cultures to more rapidly ferment carbon sources other than glucose, illustrating the functional consequence of this transcriptional adaptation (53).…”