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

ABSTRACT

Bacteria have remarkable mechanisms to survive severe external stresses and one of the most enigmatic is the non-replicative persistent (NRP) state. Practically, NRP bacteria are difficult to treat, so inhibiting proteins underlying this survival state may render such bacteria more susceptible to external stresses including antibiotics. Unfortunately, we know little about the proteins and mechanisms conferring survival through NRP. Here, we report a universal stress protein (Usps) is a primary regulator of bacterial survival through the NRP state in Micrococcus luteus NCTC 2665 (M. luteus), a BSL-1 actinobacterium. Usps are widely conserved and many bacteria including M. tuberculosis, M. smegmatis, and E. coli have multiple paralogs with overlapping functions that have obscured their functional roles. A kanamycin resistance cassette inserted into the M. luteus Universal Stress Protein A 616 gene (ΔuspA616::kan M. luteus) ablates the UspA616 protein and drastically impairs M. luteus survival under even short-term starvation (%Survival: 83% WT versus 32% ΔuspA616::kan M. luteus) and hypoxia (%Survival: 96% WT versus 48% ΔuspA616::kan M. luteus). We observed no detrimental UspA616 knockout phenotype in logarithmic growth. Proteomics demonstrated statistically significant log-phase upregulation of glyoxylate pathway enzymes isocitrate lyase and malate synthase in ΔuspA616::kan M. luteus. We note that these enzymes and the M. tuberculosis UspA616 homolog (Rv2623) are important in M. tuberculosis virulence and chronic infection, suggesting Usps are important stress proteins across diverse bacterial species. We propose that UspA616 is a metabolic switch that controls survival by regulating the glyoxylate shunt.

IMPORTANCE Bacteria tolerate severe external stresses including antibiotics through a non-replicative persistent (NRP) survival state, yet the proteins regulating this survival state are largely unknown. We show a specific universal stress protein (UspA616) controls NRP in Micrococcus luteus. Usps are widely conserved across bacteria, but their biological function(s) have remained elusive. UspA616 inactivation renders M. luteus susceptible to stress: bacteria die instead of adapting through NRP. UspA616 regulates malate synthase and isocitrate lyase, glyoxylate pathway enzymes important for chronic M. tuberculosis infection. These data show that UspA616 regulates NRP stress survival in M. luteus and suggests a function for homologous proteins in other bacteria. Importantly, inhibitors of UspA616 and homologs may render NRP bacteria more susceptible to stresses including current antibiotics.