A novel gene amplification causes upregulation of the PatAB ABC transporter and fluoroquinolone resistance in Streptococcus pneumoniae

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.

FIG 1

Expression of patA and patB and accumulation of ethidium bromide in M184 and R6^M184 transformants. (A) Expression of patA (black bars) and patB (gray bars) relative to that of rpoB. (B) Accumulation of ethidium bromide after 10 min of incubation with 100 μM ethidium bromide. *, accumulation significantly lower than that of R6 (P < 0.05).

FIG 2

Evidence of genomic rearrangements from improperly paired reads. (a) Depth of reads aligned to the hexA and patA region following removal of properly paired reads by filtering for M184 (solid line) and R6 (dashed line). The y axis indicates read depth of improperly placed reads. (b) Matches found by a BLAST search between a contig assembled from improperly paired reads and the R6 genome.

FIG 3

Evidence of duplication of a genomic region, including patA and patB, from comparison of per gene normalized read depths. (a) Per gene RPKM values for M184 plotted against those for R6. Six genes (filled circles) appeared to be present at a higher copy number than the remaining genes (gray crosses) in M184 than in R6. On further inspection, these genes were contiguous and included patA and patB (indicated). The hexA gene immediately upstream of patA (filled triangle) was not included in the higher-copy-number region. (b) The same six genes were present at increased copy numbers in M184 and the three R6^M184 transformants (T1 to T3). The slope of the regression line calculated for the high-copy-number genes (upper) was divided by the slope of the regression line for the remainder of the genome (lower) to obtain an estimate of the copy number of the amplified genes.

FIG 4

Measurement of expression of genes contained within the duplicated region of M184. (A) Representation of the genomic region around patA and patB. Dotted lines represent the extent of the duplicated region in M184 and the three transformants. (B) Expression of seven genes contained within the duplication measured by qRT-PCR from R6 (black bars) and M184 (white bars). Error bars represent the standard deviations from three biological replicates.

FIG 5

Measurement of activity of the patA promoter with and without the tRNA gene found upstream of patA copy 2 in strains containing the duplication. (A) Representation of the cloning of the tRNA gene and patA promoter upstream of a promoterless gfp gene in vector pBAV1K2. (B) Fluorescence levels measured from R6 cells containing pBAV1K2 (Empty vector), pBAV1K2p (p), and pBAV1K2tp (tp). Error bars represent the standard deviations from three biological replicates. *, fluorescence significantly greater than that from pBAV1K2p (P < 0.05, one-tailed Student's t test).