A color-based competition assay for studying bacterial stress responses in Micrococcus luteus

Abstract

Competition assays measure differences between populations of bacteria after stress adaptation, populations of different bacteria and mutations in antibiotic resistance genes. We have developed a competition-based assay to evaluate if genes upregulated under starvation are important for bacterial survival. Stress responses are critical for survival in non-pathogenic and pathogenic bacteria alike including Mycobacterium tuberculosis, Enterococcus fecaelis, Escherichia coli and Staphylococcus aureus. Unfortunately, most stress-survival proteins are poorly understood because suitable model bacteria and techniques are limited. To address this problem, we have engineered Micrococcus luteus NCTC 2665 (M. luteus) for competition assays by inactivating the sarcinaxanthin biosynthesis gene crtE (ΔcrtE), changing M. luteus colonies from yellow to white. This change allows easy identification in mixed cultures. The crtE knockout is relatively neutral for growth in complex and minimal acetate media and shows a measured fitness of one in competition with yellow wild-type bacteria. The ΔcrtE M. luteus competition assay identified a competition defect in a M. luteus strain when a specific universal stress protein was inactivated, suggesting a negative survival phenotype for this protein. We anticipate this competition assay can identify defects in other gene knockouts and mutational studies in M. luteus and will enhance our understanding of bacterial survival mechanisms.

A Universal Stress Protein That Controls Bacterial Stress Survival in Micrococcus luteus

“…These data demonstrate that ΔuspA616::kan M. luteus has a severe defect in survival under starvation and corroborate our published competition experiments (30). usp knockout experiments in E. coli or M. tuberculosis generated only modest in vitro phenotypes, but we observed robust in vitro phenotypes for ΔuspA616::kan M. luteus that correlated with the in vivo phenotype for Rv2623 knockout in M. tuberculosis (15,21,31).…”

“…High levels of both isocitrate lyase and malate synthase in early logarithmic phase would enhance growth in AMM compared to that of wild-type bacteria ( Fig. 2a and c) (30). Critically, isocitrate lyase was retained and malate synthase was lost after acetate was depleted at day 5.…”

“…Control for genetic manipulation in bacterial stress experiments. We previously evaluated knockout of the crtE carotenoid gene in M. luteus for developing a colorimetric fitness assay (30). Genetic manipulations and the pUCK4 kanamycin resistance cassette were identical for insertional inactivation of the uspA616 and crtE genes, respectively.…”

“…Nonreplicative persistent state. Our initial experiments in Micrococcus luteus were focused on understanding this important bacterial survival mechanism in one of only few bacterial species with a well-defined and reproducible NRP state (22,30). Persistence was first observed by failure of penicillin to sterilize Staphylococcus aureus cultures (25).…”

Comparative Genomics Reveals Broad Genetic Diversity, Extensive Recombination and Nascent Ecological Adaptation in Micrococcus luteus

“…The history of knowledge of M. luteus could date back to 1922, when Fleming used it to prove the lytic activity of lysozyme found in body secretions and tissues [4,5]. Since then, M. luteus was gradually used in a variety of scienti c elds [6][7][8][9][10]. One of the most famous application is resuscitating and stimulating the protein called resuscitation-promoting factor (Rpf), which could promote the resuscitation and growth of some bacteria by autocrine and paracrine signalling functions (e.g., Mycobacterium, Arthrobacter, Bacillus, Streptomyces, etc) [14].…”