In Bacillus subtilis, the SatA (Formerly YyaR) Acetyltransferase Detoxifies Streptothricin via Lysine Acetylation

Abstract

Soil is a complex niche, where survival of microorganisms is at risk due to the presence of antimicrobial agents. Many microbes chemically modify cytotoxic compounds to block their deleterious effects. Streptothricin is a broad-spectrum antibiotic produced by streptomycetes that affects Gram-positive and Gram-negative bacteria alike. Here we identify the SatA (for treptothricinceyltransferase , formerly YyaR) enzyme of as the mechanism used by this soil bacterium to detoxify streptothricin. strains lacking were susceptible to streptothricin. Ectopic expression of restored streptothricin resistance to (SatA) strains. Purified SatA acetylated streptothricin at the expense of acetyl-coenzyme A (acetyl-CoA). A single acetyl moiety transferred onto streptothricin by SatA blocked the toxic effects of the antibiotic. SatA bound streptothricin with high affinity ( [dissociation constant] = 1 μM), and did not bind acetyl-CoA in the absence of streptothricin. Expression of in conferred streptothricin resistance, indicating that SatA was necessary and sufficient to detoxify streptothricin. Using this heterologous system, we showed that the SatA homologue from also had streptothricin acetyltransferase activity. Our data highlight the physiological relevance of lysine acetylation for the survival of in the soil. Experimental support is provided for the functional assignment of gene products of the soil-dwelling bacilli and This study focuses on one enzyme that is necessary and sufficient to block the cytotoxic effects of a common soil antibiotic. The enzyme alluded to is a member of a family of proteins that are broadly distributed in all domains of life but poorly studied in and The initial characterization of the enzyme provides insights into its mechanism of catalysis.

Insights into the Function of the N -Acetyltransferase SatA That Detoxifies Streptothricin in Bacillus subtilis and Bacillus anthracis

“…The oligomeric state of all the purified BsSatA variants was determined by size exclusion chromatography using fast protein liquid chromatography (FPLC). Previously, we showed that BsSatA WT forms dimers in solution (26). The retention time of all B. subtilis BsSatA variants was consistent with a dimeric state ( Table 1), suggesting that the impaired ability to confer streptothricin resistance in S. enterica by the BsSatA variants was not due to a block in dimerization.…”

“…These results suggested that the residues L128 and A137 play a role in protein folding, oligomerization, substrate binding, or catalysis. In previous studies, we showed that S. enterica strains that synthesize BsSatA WT are resistant to 10 M streptothricin without the addition of inducer (26), indicating that the P BAD promoter of pCV1 allows for enough residual expression of satA in the absence of arabinose to confer resistance. Importantly, adding arabinose to the medium (200 M) did not overcome the inhibitory effect of streptothricin when the tester strain synthesized either the BsSatA L128F or BsSatA A137T variant ( Fig.…”

“…Changes exerted by different side chains at a specific location were analyzed, with emphasis on the binding of streptothricin to the variants. The functionality of the variants was assessed using Salmonella enterica, an enterobacterium that is naturally susceptible to streptothricin (26) and provides a genetically tractable model system for understanding what is necessary and sufficient for streptothricin resistance.…”

Staphylococcus aureus modulates the activity of acetyl‐Coenzyme A synthetase (Acs) by sirtuin‐dependent reversible lysine acetylation

“…The clarified cell‐free extract was loaded onto a 5‐mL Ni‐NTA affinity column (HisPur; ThermoFisher Scientific) and purified as described (Burckhardt and Escalante‐Semerena, ). Briefly, the column was washed with 10 bed volumes of binding buffer followed by six bed volumes of wash buffer [HEPES (50mM, pH 7.5) + NaCl (500 mM) + imidazole (40 mM)].…”

“…Many of these acetyltransferases belong to the GCN5‐related N ‐acetyltransferase (GNAT) protein superfamily (PF00583), all sharing a core catalytic domain despite low sequence homology (Vetting et al , ). GNATs are found in all domains of life and transfer an acetyl group from acetyl‐coenzyme A (AcCoA) to amines of small molecules or the amino group ( Nα ) of the N terminus of proteins, or Nε of lysyl residues in proteins or small molecules (Tucker and Escalante‐Semerena, ; Burckhardt and Escalante‐Semerena, ). Acetylation of proteins can impact their stability, structure or activity (Walsh et al , ).…”