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

Abstract

Acylation of epsilon amino groups of lysyl side chains is a widespread modification of proteins and small molecules in cells of all three domains of life. Recently, we showed that Bacillus subtilis and Bacillus anthracis encode the GCN5-related N-acetyltransferase (GNAT) SatA that can acetylate and inactivate streptothricin, which is a broad-spectrum antibiotic produced by actinomycetes in the soil. To determine functionally relevant residues of B. subtilis SatA (BsSatA), a mutational screen was performed, highlighting the importance of a conserved area near the C terminus. Upon inspection of the crystal structure of the B. anthracis Ames SatA (BaSatA; PDB entry 3PP9), this area appears to form a pocket with multiple conserved aromatic residues; we hypothesized this region contains the streptothricin-binding site. Chemical and site-directed mutagenesis was used to introduce missense mutations into satA, and the functionality of the variants was assessed using a heterologous host (Salmonella enterica). Results of isothermal titration calorimetry experiments showed that residue Y164 of BaSatA was important for binding streptothricin. Results of size exclusion chromatography analyses showed that residue D160 was important for dimerization. Together, these data advance our understanding of how SatA interacts with streptothricin. IMPORTANCE This work provides insights into how an abundant antibiotic found in soil is bound to the enzyme that inactivates it. This work identifies residues for the binding of the antibiotic and probes the contributions of substituting side chains for those in the native protein, providing information regarding hydrophobicity, size, and flexibility of the antibiotic binding site.

Antimicrobial Resistance Determinants Circulating among Thermophilic Campylobacter Isolates Recovered from Broilers in Ireland Over a One-Year Period

“…Aminoglycoside (ant(6)-Ib (867 bp)) and streptothricin resistance (satA) genes were also located within the multidrug resistance island (Figure 1A). Streptothricin acetyltransferase A (satA) is frequently reported in Gram positive bacilli [112] and shares less than 40% identity with the streptothricin acetyltransferase A (sat4) reported in Campylobacter [52,111,113]. Plasmid replication proteins within the MDRGI suggest a plasmid as the insertion vehicle of the resistance genes.…”

Whole Genome Sequencing and Comparative Genomic Analyses of Lysinibacillus pakistanensis LZH-9, a Halotolerant Strain with Excellent COD Removal Capability

“…Genes fosB that encoding fosfomycin resistance protein [70], tetracycline resistance genes tet(M) and tet(O) encoding paralogs of the translational GTPase, the elongation factor EF-G were present in all of the tested genomes, through which tetracycline was actively removed from the ribosome of bacteria [71,72], and phylogenetic analyses suggested that fosB genes of Lysinibacillus were likely acquired via cross-family gene exchange or HGT events (Figure S14). Genes encoding aminoglycoside adenylyltransferases that adenylated streptomycin and spectinomycin [73] were present in all of the tested genomes, except for L. contaminans DSM 25,560 and L. sphaericus OT4b.31, whereas genes satA encoding N-acetyltransferases that inactivated streptothricin via acetyl-CoA-dependent lysine acetylation [74,75] were only found in strains of L. pakistanensis and strain L. xylanilyticus t26, likely acquired via cross-order HGT (Figure S15). Genes encoding beta-lactamases involved in resistance to beta-lactam antibiotics were present in all tested genomes, except for L. contaminans DSM 25560, acquired probably via cross-class HGT from members of Clostridiales or Tissierellales (Figure S16).…”

Small-Molecule Acetylation by GCN5-Related N -Acetyltransferases in Bacteria

“…5B). In addition, a conserved aspartate (D160) was found to be necessary for dimerization (98). Together, these data help shape our understanding of how, in general, GNATs recognize and bind their ligand.…”

“…To gain insights into streptothricin binding to SatA, a detailed study of the B. anthracis SatA (BaSatA) enzyme was performed (98). The crystal structure of BaSatA displays a typical GNAT architecture with AcCoA situated in a V-shaped cleft.…”