Molecular and Metabolic Adaptations of Lactococcus lactis at Near-Zero Growth Rates

Abstract

f This paper describes the molecular and metabolic adaptations of Lactococcus lactis during the transition from a growing to a near-zero growth state by using carbon-limited retentostat cultivation. Transcriptomic analyses revealed that metabolic patterns shifted between lactic-and mixed-acid fermentations during retentostat cultivation, which appeared to be controlled at the level of transcription of the corresponding pyruvate dissipation-encoding genes. During retentostat cultivation, cells continued to consume several amino acids but also produced specific amino acids, which may derive from the conversion of glycolytic intermediates. We identify a novel motif containing CTGTCAG in the upstream regions of several genes related to amino acid conversion, which we propose to be the target site for CodY in L. lactis KF147. Finally, under extremely low carbon availability, carbon catabolite repression was progressively relieved and alternative catabolic functions were found to be highly expressed, which was confirmed by enhanced initial acidification rates on various sugars in cells obtained from near-zero-growth cultures. The present integrated transcriptome and metabolite (amino acids and previously reported fermentation end products) study provides molecular understanding of the adaptation of L. lactis to conditions supporting low growth rates and expands our earlier analysis of the quantitative physiology of this bacterium at near-zero growth rates toward gene regulation patterns involved in zerogrowth adaptation. Fundamental knowledge of microbial physiology and cellular regulation is obtained mainly from studies of microorganisms in batch cultures. However, the pace of life and its associated physiological phases in batch cultivation differ strongly from what is found in natural environments (1). During the early phase of batch cultivations, all nutrients, including carbon and energy sources, are usually present in excess and the specific growth rate of the microorganism equals the maximum specific growth rate (2). Thereby, our understanding of microbial energy metabolism originates mostly from microbial population studies performed under laboratory conditions that include rapid growth, high metabolic activity, and high cell density. However, natural microbial communities generally live under relative famine conditions with low specific growth and metabolic rates due to a limited supply of nutrients and energy sources (3). Analogously, under specific industrial fermentation conditions, microorganisms may experience strongly restricted access to nutrients for longer periods of time. For example, lactic acid bacteria (LAB) experience long periods of extremely low nutrient availability during the maturing process of dry sausage (4) and cheese (5) production. Despite these harsh conditions, several LAB survive these processes during months of maturation and may continue to contribute to flavor and aroma formation in the product matrix (4, 6, 7).Lactococcus lactis is used in food fermentation processes for several...

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

“…4A). Analogous to our earlier findings (16), many of the identified motifs appeared in a duplicated headto-tail orientation upstream of genes, which strengthens the palindromic nature of these regulatory motifs. The proposed CodY motif is present upstream of genes related to transcriptional and cell division machinery (ddl, murCDFG, mutM, papL, parC, rpmF, rexB, truA, and yacB), purine metabolic processing (guaC and purHL), and natural transformation protein (coiA, comEA, comEC, and comGC) (see Fig.…”

“…Remarkably, the previously identified highly conserved sequence element containing the palindromic 5=-CTGTCAG-3= motif that resembles the CodY binding site (Fig. 4B) (16) was also identified in profiles 3, 6, and 7 in the present study (Fig. 4A).…”

“…Induction of starvation in the high-density retentostat culture by the closing of the medium supply (0 h) is instant, which may explain the complete expression of pathways that could facilitate the import of exogenous DNA as a potential nutrient source. In this context, it is interesting that the Gram-positive bacterium L. lactis adapted to near-zero-growth conditions by extended retentostat cultivation (the state of the culture at 0 h), highly expressed the capacity to import alternative carbon sources, and was found to more rapidly utilize these carbon sources when they were provided to the cells (16). Apparently, starvation led to an expansion of this quest for alternative nutrients, including the machinery to import exogenous DNA.…”

“…The transcriptome adaptations were evaluated in the context of their regulation, where we identified a conserved cis-acting regulatory motif, and we propose a regulatory role for CodY in controlling expression of the translational machinery and natural competence. Notably, these findings suggest that CodY may play a dual role in the transcriptional adaptation to retentostat conditions (16) as well as the starvation adaptation studied here, which may involve different CodY coregulators.…”

“…Prolonged retentostat cultivation leads to growth rates that approximate zero and induces a cellular physiology in which the metabolic energy is completely invested in maintenance-associated processes (10,15), supporting high cell viability, and which is different from stationary-phase-induced starvation (10). Recent retentostat studies with L. lactis at a nearzero growth rate illustrated that lactococcal adaptation to these conditions includes the derepression of genes involved in alternative carbon source import as well as the repression of cell membrane metabolism (16). Some of these adaptations were also observed in batch cultures that entered the stationary phase of growth (17).…”