Carbohydrate starvation causes a metabolically active but nonculturable state in Lactococcus lactis

Western Dairy Center, Center for Integrated BioSystems, Center for Microbe Detection and Physiology, Department of Nutrition and Food Sciences, Utah State University, Logan, UT 84322-4700

^* To whom correspondence should be addressed. Email: bcweimer{at}cc.usu.edu.

	Abstract

This study characterized the ability of lactococci to become nonculturable under carbohydrate starvation while maintaining metabolic activity. We determined the changes in physiological parameters and extracellular substrate levels of multiple lactococcal strains in a number of environmental conditions along with whole genome expression profiles. Three distinct phases were observed - logarithmic growth, sugar exhaustion, and nonculturability. Shortly after carbohydrate starvation, the each lactococcal strain lost the ability to form colonies on solid media, but maintained an intact cell membrane and metabolic activity for over 3.5 yr. ML3, a strain that metabolized lactose rapidly, reached nonculturability within 1 wk. Other strains that metabolized lactose slowly (SK11) or not at all (IL1403) required 1-3 mon to become nonculturable. In all cases, the cells contained at least 100 pM of intracellular ATP after 6 mon of starvation and remained at that level for the remainder of the study. Aminopeptidase and lipase/esterase activities decreased below detection limits during the nonculturable phase. During sugar exhaustion and into nonculturability, serine and methionine were produced, while glutamine and arginine were depleted from the medium. The cells retained the ability to transport amino acids via PMF, and peptides by ATP-driven translocation, respectively. Addition of branched-chain amino acids to the culture medium resulted in increased intracellular ATP levels and new metabolic products, indicating that branched-chain amino acid catabolism resulted in energy and metabolic products to support survival during starvation. Gene expression analysis showed that the genes responsible for sugar metabolism were repressed as the cells entered nonculturability. The genes responsible for cell division were repressed, while autolysis and cell wall metabolism genes were neither induced at starvation nor during nonculturability. Taken together these observations verify that carbohydrate-starved lactococci attain a nonculturable state wherein sugar metabolism, cell division, and autolysis were repressed allowing the cells to maintain transcription, metabolic activity, and energy production during a state that produced new metabolites not associated with logarithmic growth.