title: Complete Reconstitution of the Vancomycin-Intermediate Staphylococcus aureus Phenotype of Strain Mu50 in Vancomycin-Susceptible S. aureus.
abstract: Complete reconstitution of the vancomycin-intermediate Staphylococcus aureus (VISA) phenotype of strain Mu50 was achieved by sequentially introducing mutations into six genes of vancomycin-susceptible S. aureus (VSSA) strain N315ΔIP. The six mutated genes were detected in VISA strain Mu50 but not in N315ΔIP. Introduction of the mutation Ser329Leu into vraS, encoding the sensor histidine kinase of the vraSR two-component regulatory (TCR) system, and another mutation, Glu146Lys, into msrR, belonging to the LytR-CpsA-Psr (LCP) family, increased the level of vancomycin resistance to that detected in heterogeneous vancomycin-intermediate S. aureus (hVISA) strain Mu3. Introduction of two more mutations, Asn197Ser into graR of the graSR TCR system and His481Tyr into rpoB, encoding the β subunit of RNA polymerase, converted the hVISA strain into a VISA strain with the same level of vancomycin resistance as Mu50. Surprisingly, however, the constructed quadruple mutant strain ΔIP4 did not have a thickened cell wall, a cardinal feature of the VISA phenotype. Subsequent study showed that cell wall thickening was an inducible phenotype in the mutant strain, whereas it was a constitutive one in Mu50. Finally, introduction of the Ala297Val mutation into fdh2, which encodes a putative formate dehydrogenase, or a 67-amino-acid sequence deletion into sle1 [sle1(Δ67aa)], encoding the hydrolase of N-acetylmuramyl-l-alanine amidase in the peptidoglycan, converted inducible cell wall thickening into constitutive cell wall thickening. sle1(Δ67aa) was found to cause a drastic decrease in autolysis activity. Thus, all six mutated genes required for acquisition of the VISA phenotype were directly or indirectly involved in the regulation of cell physiology. The VISA phenotype seemed to be achieved through multiple genetic events accompanying drastic changes in cell physiology.
authors.affiliation: Department of Microbiology, Faculty of Medicine, Juntendo University, Tokyo, Japan yukk@juntendo.ac.jp.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Katayama
authors.affiliation: Department of Microbiology, Faculty of Medicine, Juntendo University, Tokyo, Japan.
authors.forename: Miwa
authors.initials: M
authors.lastname: Sekine
authors.affiliation: Department of Microbiology, Faculty of Medicine, Juntendo University, Tokyo, Japan.
authors.forename: Tomomi
authors.initials: T
authors.lastname: Hishinuma
authors.affiliation: Research Centre for Infection Control Science, Juntendo University, Tokyo, Japan.
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Aiba
authors.affiliation: Research Centre for Infection Control Science, Juntendo University, Tokyo, Japan.
authors.forename: Keiichi
authors.initials: K
authors.lastname: Hiramatsu
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001433:Bacteriolysis / Q000235:genetics; D002473:Cell Wall / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D005560:Formate Dehydrogenases / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005838:Genotype; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D009154:Mutation; D010641:Phenotype; D059386:Reverse Genetics / Q000379:methods; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D014640:Vancomycin / Q000494:pharmacology*; D020713:Vancomycin Resistance / Q000235:genetics*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D008565:Membrane Proteins; C477297:MsrR protein, Staphylococcus aureus; C478169:VraS protein, Staphylococcus aureus; D014640:Vancomycin; D005560:Formate Dehydrogenases; D012321:DNA-Directed RNA Polymerases