The Role of Amino Acid Substitution in HepT Toward Menaquinone Isoprenoid Chain Length Definition and Lysocin E Sensitivity in Staphylococcus aureus

Abstract

Objectives: Staphylococcus aureus Smith strain is a historical strain widely used for research purposes in animal infection models for testing the therapeutic activity of antimicrobial agents. We found that it displayed higher sensitivity toward lysocin E, a menaquinone (MK) targeting antibiotic, compared to other S. aureus strains. Therefore, we further explored the mechanism of this hypersensitivity. Methods: MK production was analyzed by high-performance liquid chromatography and mass spectrometric analysis. S. aureus Smith genome sequence was completed using a hybrid assembly approach, and the MK biosynthetic genes were compared with other S. aureus strains. The hepT gene was cloned and introduced into S. aureus RN4220 strain using phage mediated recombination, and lysocin E sensitivity was analyzed by the measurement of colony-forming units. Results: We found that Smith strain produced MKs with the length of the side chain ranging between 8 and 10, as opposed to other S. aureus strains that produce MKs 7-9. We revealed that Smith strain possessed the classical pathway for MK biosynthesis like the other S. aureus. HepT, a polyprenyl diphosphate synthase involved in chain elongation of isoprenoid, in Smith strain harbored a Q25P substitution. Introduction of hepT from Smith to RN4220 led to the production of MK-10 and an increased sensitivity toward lysocin E. Conclusion: We found that HepT was responsible for the definition of isoprenoid chain length of MKs and antibiotic sensitivity.

Hybrid assembly using long reads resolves repeats and completes the genome sequence of a laboratory strain of Staphylococcus aureus subsp. aureus RN4220

“…To cover the repetitive elements while sequencing a genome, either the reads longer than the repetitive elements or alternative approaches to overcoming this problem are essential. In this study, we took advantage of our hybrid genome assembly approach [ 9 , 10 , 11 , 12 ] using long reads from ONT MinION and short reads from Illumina to complete the RN4220 genome. As high-quality long reads are much more crucial than coverage, we could complete the genome of this bacterium with low coverage long reads and high coverage short reads.…”

“…S. aureus RN4220 was routinely cultured on tryptic soy broth at 37 °C without antibiotic selection. Genome sequencing and assembly were performed as previously explained [ 9 , 10 , 11 , 12 ]. Briefly, genomic DNA was isolated from overnight culture using Qiagen DNA-blood Mini Kit (Qiagen, Hilden, Germany) and lysostaphin for bacterial lysis.…”

Hybrid assembly using ultra-long reads resolves repeats and completes the genome sequence of a laboratory strain of Staphylococcus aureus subsp. aureus RN4220

“…To cover the repetitive elements while sequencing a genome, either the reads longer than the repetitive elements or alternative approaches to overcoming this problem are essential. In this study, we took the advantage of our hybrid genome assembly approach[7–10] using long reads from ONT MinION and short reads from Illumina to complete the RN4220 genome. As high-quality long reads are much crucial than coverage, we could complete the genome of this bacterium with low coverage long reads, and high coverage short reads.…”

“…S. aureus RN4220 was routinely cultured on tryptic soy broth at 37°C without antibiotic selection. Genome sequencing and assembly was performed as previously explained[7–10]. Briefly, genomic DNA was isolated from overnight culture using Qiagen DNA-blood Mini Kit (Qiagen, Hilden, Germany) and lysostaphin for bacterial lysis.…”

High prevalence of heteroresistance in Staphylococcus aureus is caused by a multitude of mutations in core genes

“…In summary, for S . aureus HR isolates the dominance of single-nucleotide polymorphisms and small insertions/deletions in generating resistance [ 50 , 51 , 91 – 93 ], and the absence of gene amplification, can be explained by a combination of a generally low content of resistance genes and absence of surrounding repeat sequences as compared to gram-negative species.…”