PhoU2 but Not PhoU1 as an Important Regulator of Biofilm Formation and Tolerance to Multiple Stresses by Participating in Various Fundamental Metabolic Processes in Staphylococcus epidermidis

Abstract

PhoU, a conserved protein that has been proposed to coordinate phosphate import, is a negative regulator of drug tolerance in most bacteria. In <i>Staphylococcus epidermidis</i>, the role of PhoU in biofilm formation and drug tolerance has not yet been investigated. Two PhoU homologs in the genome of <i>S. epidermidis</i> have been identified by the presence of the conserved motif E(D)XXXD of PhoU. We separately constructed Δ<i>phoU1</i> and Δ<i>phoU2</i> mutants of <i>S. epidermidis</i> strain 1457. The Δ<i>phoU2</i> mutant displayed growth retardation, a weakened biofilm formation capacity, a higher sensitivity to H₂O₂, and reduced tolerance to multiple antibiotics. However, deletion of <i>phoU1</i> had no effect on those. We compared the transcriptome profiles of the Δ<i>phoU2</i> and Δ<i>phoU1</i> mutants with that of the parent strain. In the Δ<i>phoU2</i> mutant, expression of genes related to inorganic phosphate uptake was significantly upregulated (<i>pst</i> operon) and the levels of intracellular inorganic polyphosphate (polyP) were increased. In the Δ<i>phoU2</i> mutant, expression of enzymes in the pentose phosphate pathway (PPP) was downregulated and less NADP (NADPH) was detected, consistent with the high sensitivity to H₂O₂ and the growth retardation of the Δ<i>phoU2</i> mutant. The upregulated expression of ATP synthase was consistent with the high intracellular ATP content in the Δ<i>phoU2</i> mutant, which may have been related to the lower drug tolerance of the Δ<i>phoU2</i> mutant. This study demonstrates that PhoU2, but not PhoU1, in <i>S. epidermidis</i> regulates bacterial growth, biofilm formation, oxidative stress, and drug tolerance in association with alterations to inorganic phosphate metabolism, the pentose phosphate pathway, galactose metabolism, the tricarboxylic acid (TCA) or citric cycle, glycolysis and gluconeogenesis, and respiratory reactions.<b>IMPORTANCE</b> PhoU is widely conserved throughout the bacterial kingdom and plays an important role in response to stress and metabolic maintenance. In our study, two PhoU homologs were found in <i>S. epidermidis</i> The function of <i>phoU2</i>, but not <i>phoU1</i>, in <i>S. epidermidis</i> is related to growth, drug tolerance, the oxidative stress response, polyP levels, and ATP accumulation. In addition, <i>phoU2</i> regulates biofilm formation. Hence, <i>phoU2</i> is a regulator of both drug tolerance and biofilm formation, which are two bacterial properties that present major challenges to the clinical treatment of infections. Analysis of differential gene expression revealed that <i>phoU2</i> is involved in fundamental metabolic processes, such as the PPP pathway. These findings indicate that <i>phoU2</i> is a crucial regulator in <i>S. epidermidis</i>.