Altered lipid composition in Streptococcus pneumoniae cpoA mutants

Abstract

Background: Penicillin-resistance in Streptococcus pneumoniae is mainly due to alterations in genes encoding the target enzymes for beta-lactams, the penicillin-binding proteins (PBPs). However, non-PBP genes are altered in beta-lactam-resistant laboratory mutants and confer decreased susceptibility to beta-lactam antibiotics. Two piperacillin resistant laboratory mutants of Streptococcus pneumoniae R6 contain mutations in the putative glycosyltransferase gene cpoA. The CpoA gene is part of an operon including another putative glycosyltransferase gene spr0982, both of which being homologous to glycolipid synthases present in other Gram-positive bacteria.

Results: We now show that the cpoA mutants as well as a cpoA deletion mutant are defective in the synthesis of galactosyl-glucosyl-diacylglycerol (GalGlcDAG) in vivo consistent with the in vitro function of CpoA as α-GalGlcDAG synthase as shown previously. In addition, the proportion of phosphatidylglycerol increased relative to cardiolipin in cpoA mutants. Moreover, cpoA mutants are more susceptible to acidic stress, have an increased requirement for Mg(2+) at low pH, reveal a higher resistance to lysis inducing conditions and are hypersensitive to bacitracin.

Conclusions: The data show that deficiency of the major glycolipid GalGlcDAG causes a pleitotropic phenotype of cpoA mutant cells consistent with severe membrane alterations. We suggest that the cpoA mutations selected with piperacillin are directed against the lytic response induced by the beta-lactam antibiotic.

Figure 1

Genes, transcription and deletions in the cpoA-spr0985 region of S. pneumoniae R6. (A) Wide horizontal arrows indicate genes apparently co-transcribed with cpoA (black), and flanking genes (white). spr0983.1 has not been annotated in the R6 genome [20], but its presence has been predicted from other S. pneumoniae genomes such as TIGR4 [56]. The positions and extend of in-frame deletions are shown as white boxes below the respective genes. Lines above the genetic map represent DNA products obtained by RT-PCR with total RNA and gene-specific primers. The positions of the promoter P_cpoA and of putative ρ-independent terminators (T₁ [ΔG = −10.4 kcal/mol], T₂ [ΔG = −10.1 kcal/mol]) are given by angled and vertical arrows, respectively. (B) The nucleotide sequence upstream of S. pneumoniae R6 cpoA and putative 3'-coding sequences is shown together with the predicted peptide sequence (Sp). The −10 element of P_cpoA is underlined, and the transcription start site (+1) is indicated with an angled arrow. The position of an adenine nucleotide, deleted in the mutant strain P106 [7] is marked with *Δ. Two potential start codons of the cpoA gene (ATG₁, ATG₂; see text for detail) are underlined. The respective cpoA sequences of S. mitis B6 (Sm) and S. oralis Uo5 (So) are shown below.

Figure 2

Glycolipids in ΔcpoA and piperacillin resistant laboratory mutants containing cpoA mutations. Lipids extracted from strain R6 and from cpoA mutants, P104, P106, and R6ΔcpoA as indicated above the lanes were separated by thin layer chromatography (chloroform/methanol/acetic acid = 80:15:8). GalGalDAG (S1) and GlcDAG (S2) were used as a standards. Spots were assigned to the two major glycolipids of S. pneumoniae diglycosyl DAG (GalGlcDAG) and monoglycosyl DAG (GlcDAG).

Figure 3

Phospholipids in cpoA mutants. Lipids were extracted and separated by two dimensional TLC. 1.D and 2.D: first and second dimension (first dimension: CHCl₃/MeOH/H₂0 = 65:25:4; second dimension: CHCl₃/AcOH/MeOH/H₂0 = 80:14:10:3). Phospholipids were visualized by spraying with Molybdenum Blue spray reagent. PG: phosphatidylgylcerol; CL: cardiolipin. Spots were assigned according to the phosphatidylglycerol standard (see Additional file 1: Figure S1) and Fischer [42].

Figure 4

Growth of cpoA mutants in low pH medium. Strains were grown in C-medium, and culture density was monitored by nephelometry [NU]. The growth was examined at pH 8 (circles) and pH 6 (squares). A: R6; B: P104; C: P106; D: R6ΔcpoA.

Figure 5

Mg²⁺ requirement of cpoA mutations. Strains were grown in C-medium pH 6, and culture density was monitored by nephelometry [NU]. The medium contained either 0.195 mg/ml MgCl₂ final concentration (filled circles) or 0.39 mg/ml MgCl₂ (squares). A: R6; B: P104; C: P106; D: R6ΔcpoA.

Figure 6

Triton induced lysis. Cells were grown to OD₆₀₀ in C-medium. At OD₆₀₀ = 0.5, Triton (0.01% final concentration) was added. R6: filled circles; R6ΔcpoA: open circles; P106: open triangles; P104: open squares.