Insertional inactivation of branched-chain alpha-keto acid dehydrogenase in Staphylococcus aureus leads to decreased branched-chain membrane fatty acid content and increased susceptibility to certain stresses

Abstract

Staphylococcus aureus is a major community and nosocomial pathogen. Its ability to withstand multiple stress conditions and quickly develop resistance to antibiotics complicates the control of staphylococcal infections. Adaptation to lower temperatures is a key for the survival of bacterial species outside the host. Branched-chain alpha-keto acid dehydrogenase (BKD) is an enzyme complex that catalyzes the early stages of branched-chain fatty acid (BCFA) production. In this study, BKD was inactivated, resulting in reduced levels of BCFAs in the membrane of S. aureus. Growth of the BKD-inactivated mutant was progressively more impaired than that of wild-type S. aureus with decreasing temperature, to the point that the mutant could not grow at 12 degrees C. The growth of the mutant was markedly stimulated by the inclusion of 2-methylbutyrate in the growth medium at all temperatures tested. 2-Methylbutyrate is a precursor of odd-numbered anteiso fatty acids and bypasses BKD. Interestingly, growth of wild-type S. aureus was also stimulated by including 2-methylbutyrate in the medium, especially at lower temperatures. The anteiso fatty acid content of the BKD-inactivated mutant was restored by the inclusion of 2-methylbutyrate in the medium. Fluorescence polarization measurements indicated that the membrane of the BKD-inactivated mutant was significantly less fluid than that of wild-type S. aureus. Consistent with this result, the mutant showed decreased toluene tolerance that could be increased by the inclusion of 2-methylbutyrate in the medium. The BKD-inactivated mutant was more susceptible to alkaline pH and oxidative stress conditions. Inactivation of the BKD enzyme complex in S. aureus also led to a reduction in adherence of the mutant to eukaryotic cells and its survival in a mouse host. In addition, the mutant offers a tool to study the role of membrane fluidity in the interaction of S. aureus with antimicrobial substances.

FIG. 1.

(A) Schematic organization of the bkd gene cluster in three gram-positive bacteria. argR, arginine repressor; recN, DNA repair protein; ptb, phosphate acetyl/butyryltransferase family protein; bcd, leucine dehydrogenase; buk, butyrate kinase; bkdA1/bkdAA, BKD E1α subunit; bkdA2/bkdAB, BKD E1β subunit; bkdB, dihydrolipoamide acetyltransferase. bkdR encodes a SigL-dependent regulator that regulates the expression of bkd genes in B. subtilis. bkdR is absent in the cases of S. aureus and L. monocytogenes. Instead, in these species, a gene encoding a DNA repair protein (RecN) is present upstream of bkd genes. (B) Construction and confirmation of mutation in the lpd gene in S. aureus. A kanamycin resistance gene has been inserted at the HindIII site of the lpd gene. Primers P1 (5′-TATACAATCACCAGCTGCA-3′) and P2 (5′-ACAGTTATAGAAGCAGGTGA-3′) were used in PCRs that allowed amplification of a 336-bp product in the case of the genomic DNA template from wild-type S. aureus (lane 1). The same primers amplified an ∼1.8-kb amplicon in the case of an lpd mutant due to insertion of a 1.5-kb kanamycin resistance cassette (lane 3). The presence of two bands when genomic DNA from the merodiploid was used as the template suggests the presence of wild-type and mutated lpd genes (lane 2). Lane M, EcoR1/HindIII digest of λ DNA.

FIG. 2.

Growth kinetics of the wild-type S. aureus strain SH1000 and its derivative lpd mutant with and without short-chain keto acid precursors (2MB, 2-methylbutyrate; IB, isobutyrate; IV, isovalerate). Growth temperatures and the precursors used are indicated. Closed squares, wild-type S. aureus strain SH1000; open squares, wild-type SH1000 with supplement; closed triangles, lpd mutant of strain SH1000; open triangles, lpd mutant with supplement.

FIG. 3.

Growth of SH1000 and the lpd mutant on toluene overlay agar plates. SH1000 and the lpd mutant were used to inoculate BHI agar with or without 2-methylbutyrate (2-MB), and cultures were incubated at 37°C and 25°C.

FIG. 4.

Growth kinetics of the lpd mutant and its isogenic wild-type S. aureus under stress conditions. (A) Growth in BHI medium modified to pH 9.5. (B) Growth in BHI medium containing 8.8 mM H₂O₂. Closed squares, wild-type S. aureus strain SH1000; open squares, lpd mutant of strain SH1000; open triangles, lpd mutant of strain SH1000 complemented with a 5.1-kb wild-type bkd gene locus on plasmid pCU1.

FIG. 5.

(A) Autolysis of the lpd mutant (open squares) compared to autolysis of the isogenic wild-type S. aureus strain SH1000 (closed squares). (B) Autolysin profiles of freeze-thaw (lanes 1 and 2) and total (lanes 3 and 4) autolysin extracts from S. aureus strains against S. aureus 8325-4 cells. Lanes 1 and 3, wild-type S. aureus strain SH1000; lanes 2 and 4, lpd mutant of S. aureus strain SH1000. Equivalent amounts of protein samples were loaded for lane 1 and 2. Lanes 3 and 4 represent membrane-bound autolysin extracts from similar bacterial cell masses.

FIG. 6.

Survival of the lpd mutant and the isogenic wild-type S. aureus strain SH1000 in mouse. Approximately 1.15 × 10⁷ CFU (28% wild-type and 72% lpd mutant) were injected intraperitoneally into mice. Three mice were sacrificed at 4, 8, and 18 h postinjection. Fraction of lpd mutant strain (closed circles) and isogenic wild-type strain (open circles) in the injected inoculum (time zero; y axis) and in the bacteria recovered from infected liver (A) and spleen (B), respectively.