A functional dlt operon, encoding proteins required for incorporation of d-alanine in teichoic acids in gram-positive bacteria, confers resistance to cationic antimicrobial peptides in Streptococcus pneumoniae

Abstract

Streptococcus pneumoniae is one of the few species within the group of low-G +C gram-positive bacteria reported to contain no d-alanine in teichoic acids, although the dltABCD operon encoding proteins responsible for d-alanylation is present in the genomes of two S. pneumoniae strains, the laboratory strain R6 and the clinical isolate TIGR4. The annotation of dltA in R6 predicts a protein, d-alanine-d-alanyl carrier protein ligase (Dcl), that is shorter at the amino terminus than all other Dcl proteins. Translation of dltA could also start upstream of the annotated TTG start codon at a GTG, resulting in the premature termination of dltA translation at a stop codon. Applying a novel integrative translation probe plasmid with Escherichia coli 'lacZ as a reporter, we could demonstrate that dltA translation starts at the upstream GTG. Consequently, S. pneumoniae R6 is a dltA mutant, whereas S. pneumoniae D39, the parental strain of R6, and Rx, another derivative of D39, contained intact dltA genes. Repair of the stop codon in dltA of R6 and insertional inactivation of dltA in D39 and Rx yielded pairs of dltA-deficient and dltA-proficient strains. Subsequent phenotypic analysis showed that dltA inactivation resulted in enhanced sensitivity to the cationic antimicrobial peptides nisin and gallidermin, a phenotype fully consistent with those of dltA mutants of other gram-positive bacteria. In addition, mild alkaline hydrolysis of heat-inactivated whole cells released d-alanine from dltA-proficient strains, but not from dltA mutants. The results of our study suggest that, as in many other low-G+C gram-positive bacteria, teichoic acids of S. pneumoniae contain d-alanine residues in order to protect this human pathogen against the actions of cationic antimicrobial peptides.

FIG. 1.

Genetic organization of the dltA operon in S. pneumoniae R6, D39, and Rx. The organization of the dltA operon is shown in the orientation opposite to its location on the chromosome. The nucleotide sequences of the dltA promoter region and the 5′ region of the dltA open reading frame are shown. Promoter sequences (−35 and extended [ext.] −10), putative Shine-Dalgarno Sequences (SD), start and stop codons, and the transcriptional start point are shown in boldface and are underlined. The positions of the depicted sequences in the genome of S. pneumoniae R6 and the positions of the alternative start codons are indicated.

FIG. 2.

Translational fusions of dltA to E. coli ′lacZ. (A) Genetic map of the integrative translation probe plasmid pTP1. The nucleotide sequence of the multiple cloning site is shown. Authentic amino acids of β-galactosidase are highlighted in boldface. To construct translational fusions to ′lacZ, HindIII or BamHI may be used. (B) Genetic organization of the bgaA region in S. pneumoniae R6. The wild-type region of bgaA is shown, along with the same region after insertion of the translation probe plasmid pTP1. Upon integration, the endogenous bgaA gene is disrupted, and box, one of the two repetitive elements (box and rupA), is deleted. (C) Nucleotide sequence of dltA′-′lacZ fusions. Only relevant parts of the fusions are shown. The dltA promoter, which is present on all plasmids, is not shown. The Shine-Dalgarno sequence (SD) and start and stop codons are shown in boldface and are underlined. Amino acids encoded by dltA that are present in the fusion protein are underlined. Amino acids of β-galactosidase are indicated in boldface.

FIG. 3.

HPLC detection of d-alanine released from whole cells. Supernatants prepared by alkaline hydrolysis of S. pneumoniae R6 and R6dlt+ cells were derivatized with Marfey's reagent and separated by HPLC. Samples were prepared as described in Materials and Methods. The relevant part of the elution profile is shown. d-Alanine derivatives eluted at approximately 8.7 min. Their position is indicated (d-ala) and was determined by analyzing standards containing d-alanine (data not shown). mAU, milli-absorbance units.

FIG. 4.

Growth inhibition by nisin of S. pneumoniae dltA-proficient and dltA-deficient strains. Shown is the growth of isogenic strains treated with nisin concentrations determined to be the MIC of the dltA-deficient strain. The cells were grown in C medium. Cell density was monitored by nephelometry and is shown as NU. Typical growth curves to determine the MIC of nisin are shown. (A) Growth of isogenic S. pneumoniae R6 dltA pairs. Growth without added nisin is shown by open circles. Growth in the presence of a final concentration of 3 μg nisin per ml is shown by filled circles. (B) Growth of isogenic S. pneumoniae D39 dltA pairs. Growth without added nisin is shown by open circles. Growth in the presence of a final concentration of 0.6 μg nisin per ml is shown by filled circles. (C) Growth of isogenic S. pneumoniae Rx dltA pairs. Growth without added nisin is shown by open circles. Growth in the presence of a final concentration of 0.2 μg nisin per ml is shown by filled circles.