Only one of four oligopeptide transport systems mediates nitrogen nutrition in Staphylococcus aureus

Abstract

Oligopeptides internalized by oligopeptide permease (Opp) transporters play key roles in bacterial nutrition, signaling, and virulence. To date, two opp operons, opp-1 and opp-2, have been identified in Staphylococcus aureus. Systematic in silico analysis of 11 different S. aureus genomes revealed the existence of two new opp operons, opp-3 and opp-4, plus an opp-5A gene encoding a putative peptide-binding protein. With the exception of opp-4, the opp operons were present in all S. aureus strains. Within a single strain, the different opp operons displayed little sequence similarity and distinct genetic organization. Transcriptional studies showed that opp-1, opp-2, opp-3, and opp-4 operons were polycistronic and that opp-5A is monocistronic. We designed a minimal chemically defined medium for S. aureus RN6390 and showed that all opp genes were expressed but at different levels. Where tested, OppA protein production paralleled transcriptional profiles. opp-3, which encodes proteins most similar to known peptide transport proteins, displayed the highest expression level and was the only transporter to be regulated by specific amino acids, tyrosine and phenylalanine. Defined deletion mutants in one or several peptide permeases were constructed and tested for their capacity to grow in peptide-containing medium. Among the four putative Opp systems, Opp-3 was the only system able to provide oligopeptides for growth, ranging in length from 3 to 8 amino acids. Dipeptides were imported exclusively by DtpT, a proton-driven di- and tripeptide permease. These data provide a first complete inventory of the peptide transport systems opp and dtpT of S. aureus. Among them, the newly identified Opp-3 appears to be the main Opp system supplying the cell with peptides as nutritional sources.

FIG. 1.

Genetic and transcriptional organization of the S. aureus NCTC8325 opp systems. The opp genes were designated according to their homology with characterized bacterial opp genes. Gene numbers were adapted from the genomic DNA sequence annotation of S. aureus NCTC8325 (accession numbers CP000253 and NC_007795). Open reading frames adjacent to the represented loci are present on the opposite strand. Double arrows indicate the amplification products obtained during RT-PCR experiments (see Results). Primers for transcriptional analysis are listed in Table 2. P, putative promoter sequence; T, putative terminator of transcription.

FIG. 2.

Expression of opp genes and production of OppA proteins in S. aureus RN6390 grown in MM. Proteins and RNA were extracted in exponential growth phase (OD₆₀₀ of 0.5). (A) opp transcription analyzed by RT-PCR. cDNA dilutions used for transcript quantification were 1:1, 1:10, 1:100, and 1:1,000. hu transcript was used as an internal standard. (B) OppA production analyzed by Western blotting. Opp-1A, Opp-3A, and Opp-4A proteins were detected at their expected sizes of 58, 59, and 62 kDa, respectively.

FIG. 3.

Regulation of Opp-3A by the amino acid content of the medium. Proteins and RNA were extracted in exponential phase of growth (OD₆₀₀ of 0.5). (A) Opp-3A production analyzed by Western blotting. Opp-3A was detected at the expected size of 59 kDa. MM+Y, MM supplemented with Tyr; CM-F, CM depleted of Phe. (B) opp-3A transcription analyzed by RT-PCR. cDNA dilutions used for transcript quantification were 1:1, 1:10, 1:100, and 1:1,000. hu transcript was used as an internal standard. a, MM; b, MM+Y; c, CM; d, CM-F. (C) Quantification of opp-3A transcripts. Values are the means of three independent experiments with standard deviations. Transcripts were quantified using the 1:100 dilution samples. For each experiment, the amount of opp-3A transcript was given as a ratio to that of hu. (D) Quantification of Opp-3A production. Proteins were extracted in exponential growth phase in MM (OD₆₀₀ of 0.5). Values are the means of five independent determinations with standard deviations. MM+Y, MM supplemented with Tyr; MM+Y+F, MM supplemented with Tyr and Phe; MM+Y+FV, MM supplemented with Tyr and the dipeptide Phe-Val.

FIG. 4.

Peptide utilization by wild-type and opp mutant strains of S. aureus. Growth yield was calculated as the percentage of population (final OD) reached in peptide-containing medium compared to CM in the stationary phase of growth. QILQWQWL, EQVIR, and PQRF peptides were not tested on the opp-1 opp-2 opp-4 (opp-124) mutant strain. wt, wild type.

FIG. 5.

Di- and tripeptide utilization by opp-3 and dtpT mutants of S. aureus. Growth yield was calculated as the percentage of population (final OD) reached in peptide-containing medium compared to CM in the stationary phase of growth. wt, wild type.

FIG. 6.

Peptide content of the growth medium of S. aureus dtpT and opp-3 dtpT mutants. Cells were grown in CM supplemented with casein-derived peptides to the stationary phase, and peptide content of the external medium was analyzed by RP-HPLC. From bottom to top, the strains are the dtpT and opp-3 dtpT mutants. Arrows highlight main peaks that were detected at higher levels or only in the culture medium of the opp-3 dtpT mutant.