Abstract
Overexpression of the ABC transporter genes patA and patB confers efflux-mediated fluoroquinolone resistance in Streptococcus pneumoniae and is also linked to pneumococcal stress responses. Although upregulation of patAB has been observed in many laboratory mutants and clinical isolates, the regulatory mechanisms controlling expression of these genes are unknown. In this study, we aimed to identify the cause of high-level constitutive overexpression of patAB in M184, a multidrug-resistant mutant of S. pneumoniae R6. Using a whole-genome transformation and sequencing approach, we identified a novel duplication of a 9.2-kb region of the M184 genome which included the patAB genes. This duplication did not affect growth and was semistable with a low segregation rate. The expression levels of patAB in M184 were much higher than those that could be fully explained by doubling of the gene dosage alone, and inactivation of the first copy of patA had no effect on multidrug resistance. Using a green fluorescent protein reporter system, increased patAB expression was ascribed to transcriptional read-through from a tRNA gene upstream of the second copy of patAB. This is the first report of a large genomic duplication causing antibiotic resistance in S. pneumoniae and also of a genomic duplication causing antibiotic resistance by a promoter switching mechanism.
Highlights
Overexpression of the ABC transporter genes patA and patB confers efflux-mediated fluoroquinolone resistance in Streptococcus pneumoniae and is linked to pneumococcal stress responses
Due to this high prevalence, it is thought that gene amplification, rather than point mutations, provides a pool of preexisting variation allowing bacterial populations to adapt to various stresses, such as antibiotic resistance or growth on unusual carbon sources [2]
Overexpression of patAB can be transferred from M184 to R6 in a single transformation step
Summary
Overexpression of the ABC transporter genes patA and patB confers efflux-mediated fluoroquinolone resistance in Streptococcus pneumoniae and is linked to pneumococcal stress responses. Data from Salmonella suggest that, in nonselected cultures, transient duplications occur at frequencies of 10Ϫ4 to 10Ϫ2, meaning that 10% to 30% of cells in a nonselected culture have a duplication somewhere in their genome at any one time [1] Due to this high prevalence, it is thought that gene amplification, rather than point mutations, provides a pool of preexisting variation allowing bacterial populations to adapt to various stresses, such as antibiotic resistance or growth on unusual carbon sources [2]. Expression of patA and patB can be induced by several fluoroquinolones, including those that are thought not to be substrates for transport, as well as the nonquinolone DNA-damaging agent mitomycin C [8, 10] This indicates that PatAB is upregulated as part of a response to DNA damage. Overexpression of patA and patB has been shown to alleviate a fitness cost of linezolid resistance [11], and a role for PatAB in response to pH stress has been suggested [12]
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