(A) Genetic organization of smrA (left) and satAB (right) in S. suis strain ST3 (upper lane) compared to their homologs in S. pneumoniae R6 (lower lane, hatched arrows). Open reading frames are shown by arrows, with the direction of transcription indicated by the arrowhead. 1, Formamidopyrimidine-DNA glycosylase; 2, dephospho-coenzyme A kinase; 3, 50S ribosomal protein L33; 4, preprotein translocase subunit SecG; 5, exoribonuclease R; 6, UTP-glucose-1-phosphate uridylyltransferase; 7, glycerol-3-phosphate dehydrogenase; 8, satR; 9, dUTP nucleotidohydrolase; 10, phosphoglycerate mutase; 11, DNA repair protein RadA; 12, arginyl tRNA synthase (argS) repressor; 13, DNA mismatch repair protein MutS; 14, degenerate transposase; 15, glutamine amidotransferase; *, hypothetical protein; and 16, glucose-6-phosphate isomerase. Numbers between the ORFs represent the percentages of identity between their products. (B) Alignments of the promoter region of smrA and SatA and SatB from BB1001, BB1002, and BB1013. smrA, canonical −35 box and RBS are highlighted with black boxes. Two 7-bp inverted repeats (arrows) were found downstream the −35 box, separated by a 5-bp spacing region. In silico studies using Softberry Bprom software and the 133 bp immediately upstream from smrA showed that the loss of 2 base pairs in the spacer region led to the recognition of a previously undetected −10 box immediately downstream from the 3′ repeat (gray box). BB1013 bore three mutations compared to FQ-susceptible strains. G-43A is located in the 3′ inverted repeat and could interfere with regulation of smrA expression. SatA and SatB, alignment of the amino acid sequences of SatA and SatB from the three strains. Conserved residues are represented in black over a white background. Light gray and dark gray backgrounds represent one and two differences among sequences, respectively.

Levels of expression of smrA and satAB in BB1013 compared to BB1001. Hatched bars represent growth on subinhibitory concentrations of ciprofloxacin (one-fourth of the MIC of each strain). Pump expression was normalized against rpoB expression levels.

Dendrograms from the amino acid sequence of SatA and SatB and closely related homologues of streptococci and enterococci. Trees were constructed with NCBI BLAST by using Fast minimum evolution and Grishin parameters and edited using MEGA software. The bar denotes genetic distance. A clear species-dependent clustering was observed among the hits obtained. Note, however, that (i) some S. mitis, S. oralis, and S. pneumoniae strains inter-mixed and that (ii) S. sanguinis strain ATCC 49296 clusters together with viridans group streptococci, away from the 21 other members of this species represented by S. sanguinis SK49. Although this suggests a horizontal gene transfer event, it is rather a misidentification of S. sanguinis strain ATCC 49296, as its 16S rRNA gene also clusters with S. oralis and S. mitis (data not shown), pointing to SatAB as a good evolutionary marker. To clearly illustrate this species-specific grouping, only one representative of each species is shown (with the number of strains included in each branch indicated by gray numbers), except for S. mitis and S. sanguinis, for which two strains have been represented, and S. suis, for which all nonredundant hits were included. It is noteworthy that SatB yields, in S. suis, one hit less than SatA. This is due to the fact that strain 05ZYH33 has an A1056 deletion in satB that leads to a premature stop codon, rendering the identity with the query sequence too low to be represented in the tree. The similarity of both trees and the species-specific clustering suggests the coevolution of both ORFs together and with the genome and points to the nonimplication of satAB in horizontal gene transfer events.

Diffusion antibiograms of BB1013 in medium with (A) and without (B) 10 μg/ml of reserpine. CIP, ciprofloxacin; NOR, norfloxacin; LEV, levofloxacin; ENR, enrofloxacin. Numbers indicate the micrograms of antibiotic in the disc.

Protein modeling of SmrA (A and B), SatA (C), and SatB (D). (A) Lateral view of SmrA showing a 12-transmembrane-segment structure typical of the major facilitator superfamily of transporters. Arrows show the amino acid substitutions present in BB1013 compared to BB1001 and BB1002. Red- and yellow-stained residues are T107A and I126V substitutions, respectively. (B) View down the channel of SmrA. (C and D) Modeling of SatA and SatB. A six-transmembrane-segment structure followed by a nucleotide binding domain is observed in each protein.

(A) Schematic representation of smrA and satAB integration in B. subtilis. Open reading frames are shown as arrows, with the direction of transcription indicated by the arrowhead. pDR67 bears regions of homology to the nonessential amyE locus of B. subtilis. These gene fragments are shown as broken arrows. Homologous recombination thereof with the chromosome allows for pDR67 integration. smrA and satAB amplicons were ligated to pDR67 and transformed directly in B. subtilis. (B) Phenotypic screening of pDR67 integration in the chromosome. Bacteria were grown on LB containing 1% potato starch. Upper lane, B. subtilis 168 with a functional copy of amyE. Degradation of starch appears as a light unstained halo. Lower lane, BB1066, with pDR67 disrupting amyE, has no amylase activity. Starch is stained, and no light zone is visible.